Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics

ABSTRACT

Isolated polynucleotides and polypeptides encoded thereby are described, together with the use of those products for making transgenic plants with increased tolerance to abiotic stress (e.g., high or low temperature, drought, flood).

This application is a divisional of co-pending application Ser. No.11/140,450 filed on May 27, 2005, which claims priority under 35 U.S.C.§119(e) of U.S. Provisional Application No. 60/575,253 filed on May 27,2004, the entire contents of both of which are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to isolated polynucleotides, polypeptidesencoded thereby, and the use of those sequences for making transgenicplants with modulated water use efficiency.

BACKGROUND OF THE INVENTION

Plants are constantly exposed to a variety of biotic (e.g., pathogeninfection and insect herbivory) and abiotic (e.g., high or lowtemperature, drought, flood, anaerobic conditions and salinity)stresses. To survive these challenges, plants have developed elaboratemechanisms to perceive external signals and to manifest adaptiveresponses with proper physiological and morphological changes (Bohnertet al., 1995). Plants exposed to heat and/or low water or droughtconditions typically have low yields of plant material, seeds, fruit andother edible products. Some countries of the world consistently havevery low rainfall and therefore have problems growing sufficient foodcrops for their population. Yet it has been observed that some plantssurvive and thrive in low water environments. It would, therefore, be ofgreat interest and importance to be able to identify genes that conferimproved water efficiency characteristics to thereby enable one tocreate transformed plants (such as crop plants) with modulated waterefficiency characteristics to, thereby, better survive high and/or lowheat, high and/or low water, and drought or flood conditions. Exogenousapplication to plants of high concentrations of PEG and mannitol areknown to produce osmotic stress resulting in the retardation of growthand vigor and are used to assess drought responses. Exogenousapplication of ABA stimulates drought-responses in plants and can,therefore, be an important screen to identify genes that confer improvedwater efficiency.

In the field of agriculture and forestry efforts are constantly beingmade to produce plants with an increased growth potential in order tofeed the ever-increasing world population and to guarantee the supply ofreproducible raw materials. This is done conventionally through plantbreeding. The breeding process is, however, both time-consuming andlabor-intensive. Furthermore, appropriate breeding programs must beperformed for each relevant plant species.

Progress has been made in part by the genetic manipulation of plants;that is by introducing and expressing recombinant nucleic acid moleculesin plants. Such approaches have the advantage of not usually beinglimited to one plant species, but instead being transferable among plantspecies. (Zhang et al. (2004) Plant Physiol. 135:615). There is a needfor generally applicable processes that improve forest or agriculturalplant growth potential. Therefore, the present invention relates to aprocess for increasing the abiotic stress tolerance and consequently thegrowth potential in plants, characterized by expression of recombinantDNA molecules stably integrated into the plant genome.

SUMMARY OF THE INVENTION

The present invention, therefore, relates to isolated polynucleotides,polypeptides encoded thereby, and the use of those sequences for makingtransgenic plants with modulated water use efficiency.

The present invention also relates to processes for increasing thegrowth potential in plants under abnormal water conditions, recombinantnucleic acid molecules and polypeptides used for these processes andtheir uses, as well as to plants themselves.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

The following terms are utilized throughout this application:

-   Constitutive Promoter: Promoters referred to herein as “constitutive    promoters” actively promote transcription under most, but not    necessarily all, environmental conditions and states of development    or cell differentiation. Examples of constitutive promoters include    the cauliflower mosaic virus (CaMV) 35S transcript initiation region    and the 1′ or 2′ promoter derived from T-DNA of Agrobacterium    tumefaciens, and other transcription initiation regions from various    plant genes, such as the maize ubiquitin-1 promoter, known to those    of skill.-   Domain: Domains are fingerprints or signatures that can be used to    characterize protein families and/or parts of proteins. Such    fingerprints or signatures can comprise conserved (1) primary    sequence, (2) secondary structure, and/or (3) three-dimensional    conformation. Generally, each domain has been associated with either    a family of proteins or motifs. Typically, these families and/or    motifs have been correlated with specific in-vitro and/or in-vivo    activities. A domain can be any length, including the entirety of    the sequence of a protein. Detailed descriptions of the domains,    associated families and motifs, and correlated activities of the    polypeptides of the instant invention are described below. Usually,    the polypeptides with designated domain(s) can exhibit at least one    activity that is exhibited by any polypeptide that comprises the    same domain(s).-   Drought: Plant species vary in their capacity to tolerate drought    conditions. For each species, optimal growth can be achieved if a    certain level of water is always available. Other factors such as    temperature and soil conditions have a significant impact on the    availability of water to the plant. “Drought” can be defined as the    set of environmental conditions under which a plant will begin to    suffer the effects of water deprivation, such as decreased    photosynthesis, loss of turgor (wilting) and decreased stomatal    conductance. This drought condition results in a significant    reduction in yield. Water deprivation may be caused by lack of    rainfall or limited irrigation. Alternatively, water deficit may    also be caused by high temperatures, low humidity, saline soils,    freezing temperatures or water-logged soils that damage roots and    limit water uptake to the shoot. Since plant species vary in their    capacity to tolerate water deficit, the precise environmental    conditions that cause drought stress can not be generalized.    However, drought tolerant plants produce higher biomass and yield    than plants that are not drought tolerant. Differences in physical    appearance, recovery and yield can be quantified and statistically    analyzed using well known measurement and analysis methods.-   Endogenous: The term “endogenous,” within the context of the current    invention refers to any polynucleotide, polypeptide or protein    sequence which is a natural part of a cell or organisms regenerated    from said cell.-   Exogenous: “Exogenous,” as referred to within, is any    polynucleotide, polypeptide or protein sequence, whether chimeric or    not, that is initially or subsequently introduced into the genome of    an individual host cell or the organism regenerated from said host    cell by any means other than by a sexual cross. Examples of means by    which this can be accomplished are described below, and include    Agrobacterium-mediated transformation (of dicots—e.g. Salomon et al.    EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO J. 2:987 (1983);    of monocots, representative papers are those by Escudero et al.,    Plant J. 10:355 (1996), Ishida et al., Nature Biotechnology 14:745    (1996), May et al., Bio/Technology 13:486 (1995)), biolistic methods    (Armaleo et al., Current Genetics 17:97 1990)), electroporation, in    planta techniques, and the like. Such a plant containing the    exogenous nucleic acid is referred to here as a T₀ for the primary    transgenic plant and T₁ for the first generation. The term    “exogenous” as used herein is also intended to encompass inserting a    naturally found element into a non-naturally found location.-   Flood: Plant species vary in their capacity to tolerate flooding.    Some plants, such as rice, are cultivated in water while plants such    as corn do not tolerate flooding. “Flood,” as referred to within, is    the state of water saturation at which soils become hypoxic or    anoxic, thus limiting respiration in the root. Reduced respiration    damages roots and can limit the permeability of roots to water,    resulting in decreased leaf water potential and wilting. Since plant    species vary in their capacity to tolerate flooding, the precise    environmental conditions that cause flood stress can not be    generalized. However, flood tolerant plants are characterized by    their ability to retain their normal appearance or recover quickly    from flood. Such flood tolerant plants produce higher biomass and    yield than plants that are not flood tolerant. Differences in    physical appearance, recovery and yield can be quantified and    statistically analyzed using well known measurement and analysis    methods.-   Functionally Comparable Proteins: This phrase describes those    proteins that have at least one characteristic in common. Such    characteristics include sequence similarity, biochemical activity,    transcriptional pattern similarity and phenotypic activity.    Typically, the functionally comparable proteins share some sequence    similarity or at least one biochemical and within this definition,    homologs, orthologs and analogs are considered to be functionally    comparable. In addition, functionally comparable proteins generally    share at least one biochemical and/or phenotypic activity.

Functionally comparable proteins will give rise to the samecharacteristic to a similar, but not necessarily to the same degree.Typically, comparable proteins give the same characteristics where thequantitative measurement due to one of the comparables is at lest 20% ofthe other; more typically, between 30 to 40%; even more typically,between 50-60%; even more typically, 70 to 80%; even more typicallybetween 90 to 100%.

-   Heterologous sequences: “Heterologous sequences” are those that are    not operatively linked or are not contiguous to each other in    nature. For example, a promoter from corn is considered heterologous    to an Arabidopsis coding region sequence. Also, a promoter from a    gene encoding a growth factor from corn is considered heterologous    to a sequence encoding the corn receptor for the growth factor.    Regulatory element sequences, such as UTRs or 3′ end termination    sequences that do not originate in nature from the same gene as the    coding sequence originates from, are considered heterologous to said    coding sequence. Elements operatively linked in nature and    contiguous to each other are not heterologous to each other. On the    other hand, these same elements remain operatively linked but become    heterologous if other filler sequence is placed between them. Thus,    the promoter and coding sequences of a corn gene expressing an amino    acid transporter are not heterologous to each other, but the    promoter and coding sequence of a corn gene operatively linked in a    novel manner are heterologous.-   High Temperature: Plant species vary in their capacity to tolerate    high temperatures. Very few plant species can survive temperatures    higher than 45° C. The effects of high temperatures on plants,    however, can begin at lower temperatures depending on the species    and other environmental conditions such as humidity and soil    moisture. “High temperature” can be defined as the temperature at    which a given plant species will be adversely affected as evidenced    by symptoms such as decreased photosynthesis. Since plant species    vary in their capacity to tolerate high temperature, the precise    environmental conditions that cause high temperature stress can not    be generalized. However, high temperature tolerant plants are    characterized by their ability to retain their normal appearance or    recover quickly from high temperature conditions. Such high    temperature tolerant plants produce higher biomass and yield than    plants that are not high temperature tolerant. Differences in    physical appearance, recovery and yield can be quantified and    statistically analyzed using well know measurement and analysis    methods.-   Inducible Promoter: An “inducible promoter” in the context of the    current invention refers to a promoter which is regulated under    certain conditions, such as light, chemical concentration, protein    concentration, conditions in an organism, cell, or organelle, etc. A    typical example of an inducible promoter, which can be utilized with    the polynucleotides of the present invention, is PARSK1, the    promoter from the Arabidopsis gene encoding a serine-threonine    kinase enzyme, and which promoter is induced by dehydration,    abscissic acid and sodium chloride (Wang and Goodman, Plant J. 8:37    (1995)). Examples of environmental conditions that may affect    transcription by inducible promoters include anaerobic conditions,    elevated temperature, or the presence of light.-   Low Temperature: Plant species vary in their capacity to tolerate    low temperatures. Chilling-sensitive plant species, including may    agronomically important species, can be injured by cold,    above-freezing temperatures. At temperatures below the    freezing-point of water most plant species will be damaged. Thus,    “low temperature” can be defined as the temperature at which a given    plant species will be adversely affected as evidenced by symptoms    such as decreased photosynthesis and membrane damage (measured by    electrolyte leakage). Since plant species vary in their capacity to    tolerate low temperature, the precise environmental conditions that    cause low temperature stress can not be generalized. However, low    temperature tolerant plants are characterized by their ability to    retain their normal appearance or recover quickly from low    temperature conditions. Such low temperature tolerant plants produce    higher biomass and yield than plants that are not low temperature    tolerant. Differences in physical appearance, recovery and yield can    be quantified and statistically analyzed using well known    measurement and analysis methods.

Plant seeds vary considerably in their ability to germinate under lowtemperature conditions. Seeds of most plant species will not germinateat temperatures less than 10° C. Once seeds have imbibed water theybecome very susceptible to disease, water and chemical damage. Seedsthat are tolerant to low temperature stress during germination cansurvive for relatively long periods under which the temperature is toolow to germinate. Since plant species vary in their capacity to toleratelow temperature during germination, the precise environmental conditionsthat cause low temperature stress during germination can not begeneralized. However, plants that tolerate low temperature duringgermination are characterized by their ability to remain viable orrecover quickly from low temperature conditions. Such low temperaturetolerant plants produce, germinate, become established, grow morequickly and ultimately produce more biomass and yield than plants thatare not low temperature tolerant. Differences in germination rate,appearance, recovery and yield can be quantified and statisticallyanalyzed using well known measurement and analysis methods.

-   Masterpool: The “master pools” discussed in these experiments are a    pool of seeds from five different transgenic plants transformed with    the same exogenous gene.-   Misexpression: The term “misexpression” refers to an increase or a    decrease in the transcription of a coding region into a    complementary RNA sequence as compared to the wild-type. This term    also encompasses expression of a gene or coding region for a    different time period as compared to the wild-type and/or from a    non-natural location within the plant genome.-   Percentage of sequence identity: “Percentage of sequence identity,”    as used herein, is determined by comparing two optimally aligned    sequences over a comparison window, where the fragment of the    polynucleotide or amino acid sequence in the comparison window may    comprise additions or deletions (e.g., gaps or overhangs) as    compared to the reference sequence (which does not comprise    additions or deletions) for optimal alignment of the two sequences.    The percentage is calculated by determining the number of positions    at which the identical nucleic acid base or amino acid residue    occurs in both sequences to yield the number of matched positions,    dividing the number of matched positions by the total number of    positions in the window of comparison and multiplying the result by    100 to yield the percentage of sequence identity. Optimal alignment    of sequences for comparison may be conducted by the local homology    algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the    homology alignment algorithm of Needleman and Wunsch J. Mol. Biol.    48:443 (1970), by the search for similarity method of Pearson and    Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerized    implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and    TFASTA in the Wisconsin Genetics Software Package, Genetics Computer    Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.    Given that two sequences have been identified for comparison, GAP    and BESTFIT are preferably employed to determine their optimal    alignment. Typically, the default values of 5.00 for gap weight and    0.30 for gap weight length are used. The term “substantial sequence    identity” between polynucleotide or polypeptide sequences refers to    polynucleotide or polypeptide comprising a sequence that has at    least 80% sequence identity, preferably at least 85%, more    preferably at least 90% and most preferably at least 95%, even more    preferably, at least 96%, 97%, 98% or 99% sequence identity compared    to a reference sequence using the programs.

Query nucleic acid and amino acid sequences were searched againstsubject nucleic acid or amino acid sequences residing in public orproprietary databases. Such searches were done using the WashingtonUniversity Basic Local Alignment Search Tool Version 1.83 (WU-Blast2)program. The WU-Blast2 program is available on the Internet fromWashington University. A WU-Blast2 service for Arabidopsis can also befound on the internet. Typically the following parameters of WU-Blast2were used: Filter options were set to “default,” Output format was setto “gapped alignments,” the Comparison Matrix was set to “BLOSUM62,”Cutoff Score (S value) was set to “default,” the Expect (E threshold)was set to “default,” the Number of best alignments to show was set to“100,” and the “Sort output” option was set to sort the output by“pvalue,”

-   Plant Promoter: A “plant promoter” is a promoter capable of    initiating transcription in plant cells and can drive or facilitate    transcription of a nucleotide sequence or fragment thereof of the    instant invention. Such promoters need not be of plant origin. For    example, promoters derived from plant viruses, such as the CaMV35S    promoter or from Agrobacterium tumefaciens such as the T-DNA    promoters, can be plant promoters. A typical example of a plant    promoter of plant origin is the maize ubiquitin-1 (ubi-1) promoter    known to those of skill.-   Specific Promoter: In the context of the current invention,    “specific promoters” refers to promoters that have a high preference    for being active in a specific tissue or cell and/or at a specific    time during development of an organism. By “high preference” is    meant at least 3-fold, preferably 5-fold, more preferably at least    10-fold still more preferably at least 20-fold, 50-fold or 100-fold    increase in transcription in the desired tissue over the    transcription in any other tissue. Typical examples of temporal    and/or tissue specific promoters of plant origin that can be used    with the polynucleotides of the present invention, are: SH-EP from    Vigna mango and EP-C1 from Phaseolus vulgaris (Yamauchi et    al, (1996) Plant Mol Biol. 30(2)321-9.); RCc2 and RCc3, promoters    that direct root-specific gene transcription in rice (Xu et al.,    Plant Mol. Biol. 27:237 (1995) and TobRB27, a root-specific promoter    from tobacco (Yamamoto et al. (1991) Plant Cell 3:371).-   Stringency: “Stringency” as used herein is a function of probe    length, probe composition (G+C content), and salt concentration,    organic solvent concentration, and temperature of hybridization or    wash conditions. Stringency is typically compared by the parameter    T_(m), which is the temperature at which 50% of the complementary    molecules in the hybridization are hybridized, in terms of a    temperature differential from T_(m). High stringency conditions are    those providing a condition of T_(m)−5° C. to T_(m)−10° C. Medium or    moderate stringency conditions are those providing T_(m)−20° C. to    T_(m)−29° C. Low stringency conditions are those providing a    condition of T_(m)−40° C. to T_(m)−48° C. The relationship of    hybridization conditions to T_(m) (in ° C.) is expressed in the    mathematical equation    T _(m)=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)  (1)    where N is the length of the probe. This equation works well for    probes 14 to 70 nucleotides in length that are identical to the    target sequence. The equation below for T_(m) of DNA-DNA hybrids is    useful for probes in the range of 50 to greater than 500    nucleotides, and for conditions that include an organic solvent    (formamide).    T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L0.63(%    formamide)  (2)    where L is the length of the probe in the hybrid. (P. Tijessen,    “Hybridization with Nucleic Acid Probes” in Laboratory Techniques in    Biochemistry and Molecular Biology, P. C. vand der Vliet, ed., c.    1993 by Elsevier, Amsterdam.) The T_(m) of equation (2) is affected    by the nature of the hybrid; for DNA-RNA hybrids T_(m) is 10-15° C.    higher than calculated, for RNA-RNA hybrids T_(m) is 20-25° C.    higher. Because the T_(m) decreases about 1° C. for each 1% decrease    in homology when a long probe is used (Bonner et al., J. Mot Biol.    81:123 (1973)), stringency conditions can be adjusted to favor    detection of identical genes or related family members.

Equation (2) is derived assuming equilibrium and therefore,hybridizations according to the present invention are most preferablyperformed under conditions of probe excess and for sufficient time toachieve equilibrium. The time required to reach equilibrium can beshortened by inclusion of a hybridization accelerator such as dextransulfate or another high volume polymer in the hybridization buffer.

Stringency can be controlled during the hybridization reaction or afterhybridization has occurred by altering the salt and temperatureconditions of the wash solutions used. The formulas shown above areequally valid when used to compute the stringency of a wash solution.Preferred wash solution stringencies lie within the ranges stated above;high stringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

-   Superpool: As used in the context of the current invention, a    “superpool” refers to a mixture of seed from 100 different “master    pools”. Thus, the superpool contains an equal amount of seed from    500 different events, but only represents 100 transgenic plants with    a distinct exogenous nucleotide sequence transformed into them,    because the master pools are of 5 different events with the same    exogenous nucleotide sequence transformed into them.-   T₀: As used in the current application, the term “T₀” refers to the    whole plant, explant or callus tissue inoculated with the    transformation medium.-   T₁: As used in the current application, the term T₁ refers to either    the progeny of the T₀ plant, in the case of whole-plant    transformation, or the regenerated seedling in the case of explant    or callous tissue transformation.-   T₂: As used in the current application, the term T₂ refers to the    progeny of the T₁ plant. T₂ progeny are the result of    self-fertilization or cross pollination of a T₁ plant.-   T₃: As used in the current application, the term T₃ refers to second    generation progeny of the plant that is the direct result of a    transformation experiment. T₃ progeny are the result of    self-fertilization or cross pollination of a T₂ plant.    2. Important Characteristics of the Polynucleotides and Polypeptides    of the Invention

The polynucleotides and polypeptides of the present invention are ofinterest because when they are misexpressed (i.e. when expressed at anon-natural location or in an increased or decreased amount) theyproduce plants with modified water use efficiency. “Water useefficiency” is a term that includes various responses to environmentalconditions that affect the amount of water available to the plant. Forexample, under high heat conditions water is rapidly evaporated fromboth the soil and from the plant itself, resulting in a decrease ofavailable water for maintaining or initiating physiological processes.Likewise, water availability is limited during cold or droughtconditions or when there is low water content in the soil.Interestingly, flood conditions also affect the amount of wateravailable to the plant because it damages the roots and thus limits theplant's ability to transport water to the shoot. As used herein,modulating water use efficiency is intended to encompass all of thesesituations as well as other environmental situations that affect theplant's ability to use and/or maintain water effectively (e.g. osmoticstress, salinity, etc.).

The polynucleotides and polypeptides of the invention, as discussedbelow and as evidenced by the results of various experiments, are usefulfor modulating water use efficiency. These traits can be used to exploitor maximize plant products for agricultural, ornamental or forestrypurposes in different environment conditions of water supply. Modulatingthe expression of the nucleotides and polypeptides of the presentinvention leads to transgenic plants that will require less water andresult in better yield in high heat and/or drought conditions, or thathave increased tolerance levels for an excess of water and result inbetter yield in wet conditions. Both categories of transgenic plantslead to reduced costs for the farmer and better yield in theirrespective environmental conditions.

3. The Polynucleotides and Polypeptides of the Invention

The polynucleotides of the invention, and the proteins expressedthereby, are set forth in the Sequence Listing. Some of these sequencesare functionally comparable proteins.

Functionally comparable proteins are those proteins that have at leastone characteristic in common. Such characteristics can include sequencesimilarity, biochemical activity and phenotypic activity. Typically, thefunctionally comparable proteins share some sequence similarity andgenerally share at least one biochemical and/or phenotypic activity. Forexample, biochemical functionally comparable proteins are proteins thatact on the same reactant to give the same product.

Another class of functionally comparable proteins is phenotypicfunctionally comparable proteins. The members of this class regulate thesame physical characteristic, such as increased drought tolerance.Proteins can be considered phenotypic functionally comparable proteinseven if the proteins give rise to the same physical characteristic, butto a different degree.

The polypeptides of the invention also include those comprising theconsensus sequences described in Tables 1-4, 2-8, 3-9, 5-8, 6-8, 7-6,8-7, 10-6 and 11-6. A consensus sequence defines the important conservedamino acids and/or domains within a polypeptide. Thus, all thosesequences that conform to the consensus sequence are suitable for thesame purpose. Polypeptides comprised of a sequence within and defined byone of the consensus sequences can be utilized for the purposes of theinvention namely to make transgenic plants with improved water useefficiency, including improved tolerance to heat or high or low waterconditions.

4. Use of the Polynucleotides and Polypeptides to Make Transgenic Plants

To use the sequences of the present invention or a combination of themor parts and/or mutants and/or fusions and/or variants of them,recombinant DNA constructs are prepared which comprise thepolynucleotide sequences of the invention inserted into a vector, andwhich are suitable for transformation of plant cells. The construct canbe made using standard recombinant DNA techniques (Sambrook et al. 1989)and can be introduced to the species of interest byAgrobacterium-mediated transformation or by other means oftransformation as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by

-   (a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797    (1992); Hamilton et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979    (1996);-   (b) YAC: Burke et al., Science 236:806-812 (1987);-   (c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. January;    87(1):103-7 (1990);-   (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res    23: 4850-4856 (1995);-   (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et    al., J. Mol Biol 170: 827-842 (1983); or Insertion vector, e.g.,    Huynh et al., In: Glover NM (ed) DNA Cloning: A practical Approach,    Vol. 1 Oxford: IRL Press (1985); T-DNA gene fusion vectors: Walden    et al., Mol Cell Biol 1: 175-194 (1990); and-   (g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a sequence of thepresent invention with any desired transcriptional and/or translationalregulatory sequences, such as promoters, UTRs, and 3′ end terminationsequences. Vectors can also include origins of replication, scaffoldattachment regions (SARs), markers, homologous sequences, introns, etc.The vector may also comprise a marker gene that confers a selectablephenotype on plant cells. The marker typically encodes biocideresistance, particularly antibiotic resistance, such as resistance tokanamycin, bleomycin, hygromycin, or herbicide resistance, such asresistance to glyphosate, chlorosulfuron or phosphinotricin.

A plant promoter is used that directs transcription of the gene in alltissues of a regenerated plant and may be a constitutive promoter, suchas p326 or CaMV35S. Alternatively, the plant promoter directstranscription of a sequence of the invention in a specific tissue manner(tissue-specific promoter) or is otherwise under more preciseenvironmental control (inducible promoter). Various plant promoters,including constitutive, tissue-specific and inducible, are known tothose skilled in the art and can be utilized in the present invention.Typically, preferred promoters to use in the present invention are thosethat are induced by heat or low water conditions Such as the RD29apromoter (Kasuga et al., Plant Cell Physiol. 45:346 (2004) andYamaguchi-Shinozaki and Shinozaki, Mol Gen Genet. 236: 331 (1993)) orother DRE-containing (dehydration-responsive elements) promoters (Liu etal, Cell 10: 1391 (1998)). Another preferred embodiment of the presentinvention is the use of root specific promoters such as those present inthe AtXTH17, AtXTH18, AtXTH19 and AtXTH20 genes of Arabidopsis(Vissenberg et al. (2005) Plant Cell Physiol 46:192) or guard cellspecific promoters such as TGG1 or KST1 (Husebye et al. (2002) PlantPhysiol 128:1180; Plesch et al. (2001) Plant J 28:455).

Alternatively, misexpression can be accomplished using a two componentsystem, whereby the first component comprises a transgenic plantcomprising a transcriptional activator operatively linked to a promoterand the second component comprises a transgenic plant comprising asequence of the invention operatively linked to the target bindingsequence/region of the transcriptional activator. The two transgenicplants are crossed and the sequence of the invention is expressed intheir progeny. In another alternative, the misexpression can beaccomplished by transforming the sequences of the two component systeminto one transgenic plant line.

Any promoter that functions in plants can be used in the firstcomponent, such as those discussed above. Suitable transcriptionalactivator polypeptides include, but are not limited to, those encodingHAP1 and GAL4. The binding sequence recognized and targeted by theselected transcriptional activator protein (e.g. a UAS element) is usedin the second component.

Transformation

Nucleotide sequences of the invention are introduced into the genome orthe cell of the appropriate host plant by a variety of techniques. Thesetechniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g. Weising et al., Ann. Rev. Genet. 22:421 (1988); and Christou,Euphytica, v. 85, n. 1-3:13-27, (1995).

Processes for the transformation and regeneration of monocotyledonousand dicotyledonous plants are known to the person skilled in the art.For the introduction of DNA into a plant host cell a variety oftechniques is available. These techniques include transformation ofplant cells by injection (e.g. Newell, 2000), microinjection (e.g.Griesbach (1987) Plant Sci. 50 69-77), electroporation of DNA (e.g.Fromm et al. (1985) Proc. Natl Acad Sci. USA 82:5824 and Wan and Lemaux,Plant Physiol. 104 (1994), 37-48), PEG (e.g. Paszkowski et al. (1984)EMBO J. 3:2717), use of biolistics (e.g. Klein et al. (1987) Nature327:773), fusion of cells or protoplasts (Willmitzer, L., 1993Transgenic plants. In: Biotechnology, A Multi-Volume ComprehensiveTreatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds., Vol. 2,627-659, VCH Weinheim-New York-Basel-Cambridge), via T-DNA usingAgrobacterium tumefaciens (e.g. Fraley et al. (Crit. Rev. Plant. Sci. 4,1-46 and Fromm et al., Biotechnology 8 (1990), 833-844) or Agrobacteriumrhizogenes (e.g. Cho et al. (2000) Planta 210:195-204) or otherbacterial hosts (e.g. Brootghaerts et al. (2005) Nature 433:629-633), aswell as further possibilities.

In addition, a number of non-stable transformation methods well known tothose skilled in the art may be desirable for the present invention.Such methods include, but are not limited to, transient expression (e.g.Lincoln et al. (1998) Plant Mol. Biol. Rep. 16:1-4) and viraltransfection (e.g. Lacomme et al. (2001) In “Genetically EngineeredViruses” (C. J. A. Ring and E. D. Blair, Eds). Pp. 59-99, BIOSScientific Publishers, Ltd. Oxford, UK).

Seeds are obtained from the transformed plants and used for testingstability and inheritance. Generally, two or more generations arecultivated to ensure that the phenotypic feature is stably maintainedand transmitted.

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

The nucleic acids of the invention can be used to confer the trait ofincreased tolerance to heat and/or low water conditions, withoutreduction in fertility, on essentially any plant.

The nucleotide sequences according to the invention encode appropriateproteins from any organism, in particular from plants, fungi, bacteriaor animals.

The process according to the invention can be applied to any plant,preferably higher plants, pertaining to the classes of Angiospermae andGymospermae. Plants of the subclasses of the Dicotylodenae and theMonocotyledonae are particularly suitable. Dicotyledonous plants belongto the orders of the Magniolales, Laurales, Piperales Aristochiales,Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales,Hamantelidales, Eucomiales, Lenneriales, Myricales, Fagales,Casuarinales, Catyophyllales, Batales, Polygonales, Plumbaginales,Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violates,Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales,Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales,Proteates, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales,Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales,Campanulales, Rubiales, Dipsacales, and Asterales. Monocotyledonousplants belong to the orders of the Alismatales, Hydrocharitales,Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales,Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales,Cyclanthales, Pandanales, Arales, Lilliales, and Orchidales. Plantsbelonging to the class of the Gymnospermae are Pinales, Ginkgoales,Cycadales and Gnetales.

The method of the invention is preferably used with plants that areinteresting for agriculture, horticulture, biomass for bioconversionand/or forestry. Examples are tobacco, oilseed rape, sugar beet, potato,tomato, cucumber, pepper, bean, pea, citrus fruit, apple, pear, berries,plum, melon, eggplant, cotton, soybean, sunflower, rose, poinsettia,petunia, guayule, cabbage, spinach, alfalfa, artichoke, corn, wheat,rye, barley, grasses such as switch grass or turf grass, millet, hemp,banana, poplar, eucalyptus trees, conifers.

Homologs Encompassed by the Invention

Sequences of the invention include proteins comprising at least about acontiguous 10 amino acid region preferably comprising at least about acontiguous 20 amino acid region, even more preferably comprising atleast about a contiguous 25, 35, 50, 75 or 100 amino acid region of aprotein of the present invention. In another preferred embodiment, theproteins of the present invention include between about 10 and about 25contiguous amino acid region, more preferably between about 20 and about50 contiguous amino acid region, and even more preferably between about40 and about 80 contiguous amino acid region.

Due to the degeneracy of the genetic code, different nucleotide codonsmay be used to code for a particular amino acid. A host cell oftendisplays a preferred pattern of codon usage. Nucleic acid sequences arepreferably constructed to utilize the codon usage pattern of theparticular host cell. This generally enhances the expression of thenucleic acid sequence in a transformed host cell. Any of the abovedescribed nucleic acid and amino acid sequences may be modified toreflect the preferred codon usage of a host cell or organism in whichthey are contained. Modification of a nucleic acid sequence for optimalcodon usage in plants is described in U.S. Pat. No. 5,689,052.Additional variations in the nucleic acid sequences may encode proteinshaving equivalent or superior characteristics when compared to theproteins from which they are engineered.

It is understood that certain amino acids may be substituted for otheramino acids in a protein or peptide structure (and the nucleic acidsequence that codes for it) without appreciable change or loss of itsbiological utility or activity. The amino acid changes may be achievedby changing the codons of the nucleic acid sequence.

It is well known in the art that one or more amino acids in a nativesequence can be substituted with other amino acid(s), the charge andpolarity of which are similar to that of the native amino acid, i.e., aconservative amino acid substitution, resulting in a silent change.Conservative substitutes for an amino acid within the native polypeptidesequence can be selected from other members of the class to which theamino acid belongs (see below). Amino acids can be divided into thefollowing four groups: (1) acidic (negatively charged) amino acids, suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids, such as arginine, histidine, and lysine; (3) neutral polar aminoacids, such as glycine, serine, threonine, cysteine, cystine, tyrosine,asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

In a further aspect of the present invention, nucleic acid molecules ofthe present invention can comprise sequences that differ from thoseencoding a protein or fragment thereof selected from the groupconsisting of the sequences present in the Sequence Listing due to thefact that the different nucleic acid sequence encodes a protein havingone or more conservative amino acid changes.

In another aspect, biologically functional equivalents of the proteinsor fragments thereof of the present invention can have about 10 or fewerconservative amino acid changes, more preferably about 7 or fewerconservative amino acid changes, and most preferably about 5 or fewerconservative amino acid changes. In a preferred embodiment, the proteinhas between about 5 and about 500 conservative changes, more preferablybetween about 10 and about 300 conservative changes, even morepreferably between about 25 and about 150 conservative changes, and mostpreferably between about 5 and about 25 conservative changes or between1 and about 5 conservative changes.

5. Experiments Confirming the Usefulness of the Polynucleotides andPolypeptides of the Invention

5.1 Procedures

The nucleotide sequences of the invention were identified by use of avariety of screens for modified water conditions, including heat and/orlow water conditions. These screens are recognized by those skilled inthe art to be predictive of nucleotide sequences that provide plantswith improved water use efficiency including improved tolerance to heatand/or low water conditions because they emulate the differentenvironmental conditions that can result from increased heat and/or lowwater conditions, These screens generally fall into two categories (1)soil screens and (2) in vitro screens.

Soil screens have the advantage of assaying the response of the entireplant to particular conditions, such as drought or high heat. On theother hand, in vitro screens have the advantage of relying on definedmedia and so allow more defined manipulation of growth conditions.“Surrogate” in vitro screens use particular chemicals to alter the wateravailable to the plant by manipulating the concentrations and/orcomponents of the growth media. For example, the ability of the plant tomaintain the water concentration within its cells, which can occurduring times of low water in the soil, can be tested by growing plantson high sucrose media. Such a screen thus allows one to separate theeffects of water loss from roots from, for example, the water loss fromleaves during high heat conditions. Each of the screens used isdescribed in more detail below.

In general, the screens used to identify the polynucleotides andpolypeptides of the invention were conducted using superpools ofArabidopsis T₂ transformed plants. The T₁ plants were transformed with aTi plasmid containing a particular SEQ ID NO in the sense orientationrelative to a constitutive promoter and harboring the plant-selectablemarker gene phosphinothricin acetyltransferase (PAT), which confersherbicide resistance to transformed plants. For surrogate screens, seedfrom multiple superpools (1,200 T₂ seeds from each superpool) weretested. T₃ seed were collected from the resistant plants and retested onall other surrogate screens. The results of the screens conducted foreach SEQ ID NO can be found in the Examples below.

5.1.1. Mannitol

Screens for mannitol resistant seedlings are surrogate screens fordrought (see Quesada et al., Genetic analysis of salt-tolerant mutantsin Arabidopsis thaliana. Genetics. 2000 154:421-36).

Seeds are sterilized in 30% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a minimum of 3 days before use.

Manitol media is prepared by mixing 375 ml sterile 1 mM mannitol with375 ml sterile 1× MS. Approximately 1200 seeds are evenly spaced per PEGplate before incubating at 22° C. for 14 days.

Putative mannitol-resistant seedlings are transferred to MS with 3%sucrose for recovery. Approximately one week later, resistant seedlingsare transferred to soil and sprayed with Finale. Finale resistant plantsare genotyped as described below.

DNA is isolated from each plant and used in PCR reactions using thefollowing cycling conditions 95° C. for 30 sec, five cycles of 51° C.for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C.for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold.Aliquots of the reaction product are analyzed on a 1.2% agarose gelstained with ethidium bromide.

T₃ Seed from those plants containing the expected PCR product arecollected and retested on 375 mM mannitol media.

5.1.2. Polyethylene Glycol (PEG)

Screens for PEG resistant seedlings are surrogate screens for drought(see van der Week et al., Growth of Arabidopsis thaliana seedlings underwater deficit studied by control of water potential in nutrient-agarmedia. J Exp Bot. 2000 51(350):1555-62).

Seeds are sterilized in 30% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a minimum of 3 days before use.

18% PEG media is prepared by mixing 360 ml of hot sterile 50% PEG with400 ml of hot sterile 0.5× MS media. Approximately 1200 seeds are evenlyspaced per PEG plate before incubating at 22° C. for 14 days.

Putative PEG-resistant seedlings are transferred to MS with 0.01%Finale. One week later, resistant seedlings are transferred to soil.Three days later the seedlings are genotyped as described below.

DNA is isolated from each plant and used in PCR reactions using thefollowing cycling conditions 95° C. for 30 sec, five cycles of 51° C.for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C.for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold.Aliquots of the reaction product are analyzed on a 1.2% agarose gelstained with ethidium bromide.

T₃ Seed from those plants containing the expected PCR product arecollected and retested using 20% PEG media.

5.1.3. Soil Drought

Soil drought screens identify plants with enhanced tolerance to droughtand enhanced recovery after drought.

Seeds are planted in holed flats containing Zonolite vermiculite thatare placed in no-holed flats. Flats are watered with 3 L of Hoagland'ssolution and covered with a plastic dome before being placed at 4° C.After 4 days, the flats are moved to the Greenhouse (22° C.) and grown 2weeks, with 1.5 L Hoagland's solution being added every 4 days, or whentop of vermiculite is dry. The final application of Hoagland's solutionis 4 days prior to the end of the 2 weeks. After 2 weeks, 1 L Hoagland'ssolution is added to the no-holed flat. After 10 days plants are wiltedbut still green.

Green, turgid plants are transplanted to 4″ square pots containing 60%sunshine mix #5 and 40% thermo-o-rock vermiculite, with Osmocote (1tbsp/8 L) and Marathon (1 tbsp/8 L). The soil is moistened and the potssub-irrigated with water. They are grown under a plastic dome for oneday, then the plastic dome is removed for the remaining growth period.

To assess plants for enhanced recovery after drought the green wiltedplants remaining in the flats are sub-irrigated with 2.5 L Hoagland'ssolution and cover with cleared with a plastic dome. The following day,the dome is removed and green survivors transplanted to 4″ square potscontaining 60% sunshine mix #5, 40% Therm-a-rock vermiculite, Osmocote(1 tbsp/8 L) and marathon (1 tbsp/8 L). The soil is moistened and thepots sub-irrigated with water. They are grown under a plastic dome forone day, then the plastic dome is removed for the remaining growthperiod.

DNA from a leaf from each plant is transferred to FTA paper via pressureand an aliquot of the DNA containing paper used in PCR reactions usingthe following cycling conditions 94° C. for 10 min, five cycles of 94°C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, five cycles of 94°C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, 30 cycles of 94° C.for 30 sec, 53° C. for 30 sec, 72° C. for 3 min, 72° C. for 7 min and 4°C. hold. Aliquots of the reaction product are analyzed on a 1% agarosegel stained with ethidium bromide.

T₃ Seed from those plants containing the expected PCR product arecollected and retested using 50 seeds from each line.

5.1.4. Heat

High heat screens identify plants with enhanced tolerance to heat andenhanced recovery after heat.

Seeds are sterilized in 30% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a minimum of 3 days before use.

MS media, pH 5.7 is prepared. Approximately 12 seeds are evenly spacedper MS plate before incubating in the vertical position at 22° C. for 14days. Under these conditions, the plates are exposed to 12,030 LUX fromabove and 3,190 LUX from the bottom.

On day 15 the plates are transferred to a 22° C. oven, which increasedtemperature in 5° C. increments to 45° C. The duration of treatment at45° C. was based on complete and homogenous wilting of 100 wild-typeseedlings (˜10 plates). After exposure to 45° C., seedlings were placedin the horizontal position at 23° C. for recovery, where they remainedfor 4-11 days. Heat recovery was assessed based on vigor and greennessand continued growth after treatment.

Leaves of control and non-resistant plants become wilted and yellowedafter only 2 days, completely bleaching after an additional 4 days. Allwild type (WS) and non-heat resistant plants die by day 6.

DNA is isolated from each plant and used in PCR reactions using thefollowing cycling conditions 95° C. for 30 sec, five cycles of 51° C.for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C.for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold.Aliquots of the reaction product are analyzed on a 1.2% agarose gelstained with ethidium bromide.

T₃ Seed from those plants containing the expected PCR product arecollected and retested.

To differentiate between natural acquired thermo tolerance of recoveredT₃ events, seeds were sterilized and stratified in parallel to wildtypeseed that was never heat treated and wild-type controls previously heattreated with the T₂ events. 15 day old seedlings were heat shocked inthe dark at 45° C. for 5 hours, as described above. Duration oftreatment at 45° C. was based on complete and homogenous wilting ofpre-heat treated wild-type seed and un-pretreated wild-type controls.After exposure to 45° C., seedlings were returned to the permissivetemperature of 23° C. for recovery where they remained for another 7days. Thermo tolerance was assessed based on prolonged greenness andcontinued growth after treatment.

5.1.5 Heat (Soil)

Seeds are sown in pots containing soil of the following composition: 60%autoclaved Sunshine Mix #5, 40% vermiculite with 2.5 Tbsp Osmocote and2.5 Tbsp 1% granular Marathon per 25 L of soil. After sowing, pots arecovered with plastic propagation domes and seed is placed at 4° C. inthe dark for at least 3 days. Pots are then returned to the greenhouse(long day light conditions of 16 hours), covered with 55% shade clothand provided a normal watering regime.

After 7 days, seedlings were transferred to a 36° C. growth chamberunder continuous light and allowed to grow until harvest. Plants werewatered minimally so as to allow for some drying of the top soil similarto that in heat-induced drought conditions in the field.

Plants are sprayed with a mixture of 3 ml Finale in 48 oz of water.Spraying is repeated every 3-4 days until only transformants remain. Theremaining transformants were weeded to a maximum of 5 evenly spacedtransformants per pot.

T3 seed was recovered and tested for thermotolerance and recovery asdescribed above.

5.1.6 High Sucrose

Screens for germination and growth on limited nutrients and 9% sucroseare surrogate screens for the altered carbon/nitrogen balance frequentlyassociated with drought (see Laby et al., The Arabidopsissugar-insensitive mutants sis4 and sis5 are defective in abscisic acidsynthesis and response. Plant Journal 23: 587-596).

Seeds are sterilized in 30% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a minimum of 3 days before use.

MS media containing 9% sucrose is prepared. Approximately 1200 seeds areevenly spaced per MS-sucrose plate before incubating at 22° C. for 9days.

Putative sucrose-resistant green seedlings are transferred to MS plates.After one week of recovery, resistant the seedlings are genotyped asdescribed below.

DNA from a leaf from each plant is transferred to FTA paper via pressureand an aliquot of the DNA containing paper used in PCR reactions usingthe following cycling conditions 94° C. for 10 min, five cycles of 94°C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, five cycles of 94°C. for 30 sec, 60° C. for 30 sec, 72° C. for 3 min, 30 cycles of 94° C.for 30 sec, 53° C. for 30 sec, 72° C. for 3 min, 72° C. for 7 min and 4°C. hold. Aliquots of the reaction product are analyzed on a 1% agarosegel stained with ethidium bromide.

T₃ Seed from those plants containing the expected PCR product arecollected and retested using 9% sucrose MS media.

5.1.7. ABA

Screens for ABA resistant seedlings are surrogate screens for drought.

Seeds are sterilized in 30% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a minimum of 3 days before use.

MS media containing 1.5 μM ABA is prepared. Approximately 1200 seeds areevenly spaced per PEG plate before incubating at 22° C. for 14 days.

Putative ABA-resistant seedlings are transferred to MS with 0.01%Finale. One week later, resistant seedlings are transferred to soil.Three days later the seedlings are genotyped as described below.

DNA is isolated from each plant and used in PCR reactions using thefollowing cycling conditions 95° C. for 30 sec, five cycles of 51° C.for 30 sec, 72° C. for 1.15 min, 95° C. for 30 sec, 25 cycles of 48° C.for 30 sec, 72° C. for 1.15 min, 72° C. for 7 min and 4° C. hold.Aliquots of the reaction product are analyzed on a 1.2% agarose gelstained with ethidium bromide.

T₃ Seed from those plants containing the expected PCR product arecollected and retested using 1.5 μM ABA MS media.

5.1.8. Procedure for Identifying Functional Homologs and ConsensusSequences

The isolated sequence of the invention was compared to the sequencespresent in the various gene banks. Pairwise comparisons were conductedand those sequences having the highest percent identity to the querysequence identified as functional homologs.

A multi-pairwise alignment was generated using the amino acid querysequence and the amino acid sequence of the functional homologs. Thisallowed identification of the conserved regions or domains of thepolypeptide. Using the conserved regions as a guide, a consensussequence was generated. This consensus sequence indicates the criticalamino acid residues and those can be either substituted and/or deletedwithout impacting the biological function of the protein.

5.2 Results

The results of the above experiments are set forth below wherein eachindividual example relates to all of the experimental results for aparticular polynucleotide/polypeptide of the invention.

Example 1 Ceres cDNA 12331850

Clone 11830, Ceres cDNA 12331850, encodes a full-length glycosylhydrolase family 17 protein, which has similarity to elicitor induciblechitinase Nt-SubE76 GI:11071974 from (Nicotiana tabacum (C-terminalhomology only) and β-1,3-glucanase.

Ectopic expression of Ceres cDNA 12331850 under the control of theCaMV35S or 32449 promoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol, and abscissic acid (ABA).    -   Continued growth on high concentration of PEG, mannitol, and        ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12331850.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12331850 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on high PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 12331850 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (ME01297) were screened on high PEG, mannitol, andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Results:

Qualitative Analysis of 13 Superpools on PEG, Mannitol, and ABA

Resistant candidates were selected based on increased size compared tothe largest wild-type control. All three screens resulted in a decreasein germination for both wildtype and superpools as compared to seeds oncontrol media. Wild-type seeds that germinated on any of the screenswere small and never developed any first leaves even after 40 days. Toensure that even slightly tolerant individuals were not omitted,seedlings that showed any growth and greening whatsoever were recoveredand transferred to soil for assessment in the T₃ generation. Allrecovered candidates showed signs of vigorous re-growth on soil,although the development was slightly delayed as compared to unstressedplants, presumably because of the transient exposure to stress. Theplants transferred to soil were sprayed with Basta^(R) to eliminate anyfalse-positives, or any lines where the Basta^(R) marker was suppressed.All of the Basta^(R)-resistant candidates flowered and set seed.Resistant seedlings were recovered from Superpools 1, 7 and 11 on allscreens (Table 1-1).

TABLE 1-1 Number of Basta^(R) seedlings identified on several screens.Superpool Promoter 18% PEG 375 mM Mannitol 1.5 uM ABA SP1 35S 10 13 11SP2 35S 0 6 5 SP3 35S 0 2 10 SP4 35S 0 4 0 SP5 35S 0 0 5 SP6 35S 0 0 4SP7 35S 1 4 8 SP8 35S 0 4 1 SP9 32449 0 0 12 SP10 32449 0 0 14 SP1132449 15 3 13 SP12 32449 3 0 6 SP13 32449 0 2 1Qualitative and Quantitative Analysis of 5 Independent EventsRepresenting 35S::cDNA 12331850 on PEG, Mannitol, and ABA Screens.

Seedlings that survived transfer to soil and which wereBasta^(R)-resistant were subjected to PCR and sequencing. At least oneresistant plant in each of the three screens contained 35S::cDNA12331850 (ME01297) making this a good candidate for further testing, T₂seeds from the 5 independent transformants that contain this clone andthat were used in the pooling process were tested for Basta^(R)resistance and for stress tolerance in the 3 surrogate drought screens.

To identify two independent events of 35S::cDNA 12331850 showing PEG,mannitol and ABA resistance, 36 seedlings from each of five events,ME01297-01, 02, 03, 04, and 05 were screened as previously described.Simultaneously, Basta^(R) segregation was assessed to identify eventscontaining a single insert segregating in a 3:1 (R:S) ratio ascalculated by a chi-square test (Table 1-2). All of the eventssegregated for a single functional insert.

TABLE 1-2 Basta segregation for ME01297 individual events ProbabilityEvent Resistant Sensitive Total of Chi-test* ME01297-01 22 14 36 0.05429ME01297-02 26 10 36 0.70031 ME01297-03 25 11 36 0.44142 ME01297-04 22 1436 0.05429 ME01297-05 24 12 36 0.24821 *Chi-test to determine whetheractual ratio of resistant to sensitive differs from the expected 3:1ratio.

Events ME01297-02 and 03 were chosen as the two events because they hadthe strongest and most consistent resistance to PEG, mannitol and ABAResistance was observed for ME01297-01, 04, and 05 although not inexpected ratios in all three screens (data not shown). The controls weresown the same day and on the same plate as the individual events. Thetransgenic control in each of these plates is a segregant, from this MEline or another ME line being tested, that failed to show resistance tothe particular stress. The PEG, mannitol and ABA (Table 1-3) segregationratios observed for ME01297-02 and 03 are consistent with the presenceof a single insert, similar to what we observed for Basta resistance(Table 1-1).

On 18% PEG, the resistant seedlings Pram these two events show some rootgrowth and they are green with new leaves emerging. The mannitolresistant seedlings also showed more root and shoot growth than thesensitive seedlings. The ABA resistant seedlings showed a slightincrease in growth. The phenotype of the resistant seedlings is uniqueto each of the screens.

TABLE 1-3 Segregation of Resistance to PEG, mannitol and ABA inME01297-02 and ME01297-03 Progeny. PEG mannitol ABA ProbabilityProbability Probability R S of Chi-test R S of Chi-test R S of Chi-testME01297-02 49 23 0.174 47 25 0.057 50 22 0.276 ME01297-03 58 14 0.276 5121 0.414 56 16 0.586 Expected 54 18 54 18 54 18 (3:1 segregation)Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon High Concentrations of PEG, Mannitol, and ABA Screens.

Progeny from T₂ plants that were recovered from the three screens andwhich contained cDNA 12331850 (SP1-A1, SP1-P1, and SP1-M18) wereanalyzed and also found to be resistant to PEG, mannitol and ABAindicating that resistance is transmitted to the next generation. Takentogether, 1) the isolation of resistant seedlings containing cDNA12331850 from all three screens, 2) the inheritance of this resistancein a subsequent generation, and 3) the fact that the progeny from two ormore events from the original transformation also segregated forresistance to the stresses, provide strong evidence that cDNA 12331850when over-expressed can provide tolerance to osmotic stress.

This gene is annotated as a glycosyl hydrolase family 17, which hassimilarity to elicitor inducible chitinase Nt-SubE76 GI:11071974 fromNicotiana tabacum (C-terminal homology only). TIGR also notes that theprotein contains similarity to beta-1,3-glucanase.

Table 1-4 provides the results of the consensus sequence (SEQ ID NOs:112-120) analysis based on Ceres cDNA 12331850.

TABLE 1-4 /tmp/889111.aln

24/05/05 plur = 2.000000 -collision -box -noboxcol colbyconsensus

Example 2 Ceres cDNA 12334963

Clone 35743, Ceres cDNA 12334963, encodes a full-length putativehypothetical protein. Ectopic expression of Ceres cDNA 12334963 underthe control of the 35S promoter induces the following phenotypes;

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol, and abscissic acid (ABA).    -   Continued growth on high PEG, mannitol, and ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12334963.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12334963 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 12334963 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (ME01467) were screened on high PEG, mannitol andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Results:

Qualitative Analysis of 13 Superpools on PEG, Mannitol, and ABA.

Resistant candidates were selected based on increased size when comparedto the largest wild-type control seedlings. All three screens resultedin a decrease in germination for both wildtype and superpools comparedto seeds on control media. Wild-type seeds that germinated on any of thescreens were small and never developed any first leaves even after 40days. To ensure that even slightly tolerant individuals were notomitted, seedlings that showed any growth and greening whatsoever wererecovered and transferred to soil for assessment in the T₃ generation.All recovered candidates showed signs of vigorous re-growth on soil,although the development was slightly delayed as compared to unstressedplants, presumably because of the transient exposure to stress. Theplants transferred to soil were sprayed with Basta^(R) to eliminate anyfalse-positives, or any lines where the Basta^(R) marker was suppressed.All of the Basta^(R)-resistant candidates flowered and set seed.Resistant seedlings were recovered from Superpools 1, 7 and 11 on allscreens (Table 2-1).

TABLE 2-1 Number of stress-tolerant and Basta^(R) seedlings identifiedon drought surrogate screens. Superpool Promoter 18% PEG 375 mM Mannitol1.5 uM ABA SP1 35S 10 13 11 SP2 35S 0 6 5 SP3 35S 0 2 10 SP4 35S 0 4 0SP5 35S 0 0 5 SP6 35S 0 0 4 SP7 35S 1 4 8 SP8 35S 0 4 1 SP9 32449 0 0 12SP10 32449 0 0 14 SP11 32449 15 3 13 SP12 32449 3 0 6 SP13 32449 0 2 1Qualitative and Quantitative Analysis of 5 Independent EventsRepresenting 35S::cDNA 12334963 on PEG, Mannitol, and ABA

Seedlings that survived transfer to soil and which wereBasta^(R)-resistant were subjected to PCR and sequencing. At least oneresistant plant in each of the three osmotic screens contained 35S::cDNA12334963 (ME01467) making this a good candidate for further testing. T₂seeds from the 5 independent transformants that contain this clone andthat were used in the pooling process were tested for Basta^(R)resistance and for stress tolerance in the 3 surrogate drought screens.

To identify two independent events of 35S::cDNA 12334963 showing PEG,mannitol and ABA resistance, 36 seedlings from each of five events,ME01467-01, 02, 03, 04, and 05 were screened as previously described.Simultaneously, Basta segregation was assessed to identify eventscontaining a single insert segregating in a 3:1 (R:S) ratio ascalculated by a Chi-square test (Table 2-2). All of the lines segregatedfor a single functional insert.

TABLE 2-2 Basta^(R) segregation for ME01467 individual eventsProbability Event Resistant Sensitive Total of Chi-test* ME01467-01 28 836 0.70031 ME01467-02 29 7 36 0.44142 ME01467-03 24 12 36 0.24821ME01467-04 28 8 36 0.70031 ME01467-05 27 9 36 1 *Chi-test to determinewhether actual ratio of resistant to sensitive differs from the expected3:1 ratio.

Events ME01467-03 and 05 were chosen as the two events because had thestrongest and most consistent resistance to PEG, mannitol and ABA.Resistance was observed for ME01467-01, 02, and 04, although not inexpected ratios in all three screens (data not shown). The controls weresown the same day and in the same plate as the individual events. Thetransgenic control in each of these plates is a segregant, from this MEline or another ME line being tested, that failed to show resistance tothe particular osmotic stress. The PEG (Tables 2-3 and 2-4), mannitol(Tables 2-5 and 2-6) and ABA (Tables 2-7 and 2-8) segregation ratiosobserved for ME01467-03 and 05 are consistent with the presence of asingle insert as demonstrated by Chi-Square. This result is similar tothe observation for Basta^(R) resistance (Table 2-2).

On 18% PEG, the resistant seedlings from these two events showed someroot growth but they were also green with emergence of new leaves at day14. The mannitol-resistant seedlings showed more root and shoot growththan the PEG resistant seedlings. The resistant seedlings on ABA showthe least amount of growth, and very little root growth relative to themannitol and PEG screens. The phenotype

TABLE 2-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-03 containing 35S::cDNA 12334963 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 24 24 0 1.0 PEG Sensitive8 8 0 32 32 0of the resistant seedlings is unique on each of the screens.

TABLE 2-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-05 containing 35S::cDNA 12334963 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 26 27 0.037 0.7 PEGSensitive 10 9 0.111 36 36 0.148

TABLE 2-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-03 containing 35S::cDNA 12334963 on mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 29 27 0.148 0.4Mannitol Sensitive 7 9 0.444 36 36 0.592

TABLE 2-6 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-05 containing 35S::cDNA 12334963 on mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 18 27 3 0.0005Mannitol Sensitive 18 9 9 36 36 12

TABLE 2-7 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-03 containing 35S::cDNA 12334963 on ABA. Event Observed Expectedχ² Probability of Chi-Test ABA Resistant 25 26.25 0.0595 0.626 ABASensitive 10  8.75 0.179  35 35   0.239 

TABLE 2-8 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01467-05 containing 35S::cDNA 12334963 on ABA. Event Observed ExpectedX² Probability of Chi-Test ABA Resistant 22 27 0.926 0.054 ABA Sensitive14 9 2.78 36 36 3.706Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon Peg, mannitol and ABA

Progeny of the T₂ plants that were recovered from the three screens andwhich contained cDNA 12334963 (SP1-A12, SP1-P9, and SP1-M4) wereanalyzed and found to be resistant to PEG, mannitol and ABA, indicatingthat resistance is transmitted to the next generation.

Taken together, 1) the isolation of resistant seedlings containing cDNA12334963 from all three surrogate screens for drought, 2) theinheritance of this resistance in the next generation, and 3) the factthat the progeny from two or more events from the originaltransformation also segregated for resistance to these osmotic stresses,provides strong evidence that cDNA 12334963, when over-expressed, canprovide tolerance to drought, freezing and other osmotic stresses.

Table 2-8 provides the results of the consensus sequence (SEQ ID NOs:121-129) analysis based on Ceres cDNA 12334963.

TABLE 2-8 /tmp/Lead-clone35743.aln

23/05/05 plur = 2.000000 -collision -box -noboxcol colbyconsensus

Example 3 Ceres cDNA 12333678

Clone 26006, Ceres cDNA 12333678, encodes a full-length glycosylhydrolase.

Ectopic expression of Ceres cDNA 12333678 under the control of theCaMV35S promoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol and abscissie acid (ABA).    -   Continued growth on high PEG, mannitol and ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12333678.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12333678 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol and ABA as Surrogate Screensfor Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 12333678 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (ME01334) were screened on high PEG, mannitol andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Results:

Qualitative Analysis of 13 Superpools on PEG, Mannitol and ABA.

Resistant candidates were selected based on increased size when comparedto the largest wild-type control seedlings. All three screens resultedin a decrease in germination for both wildtype and superpools ascompared to seeds on control media. Wild-type seeds that germinated onany of the screens were small and never developed any first leaves evenafter 40 days. To ensure that even slightly tolerant individuals werenot omitted, seedlings that showed any growth and greening whatsoeverwere recovered and transferred to soil for assessment in the T₃generation. All recovered candidates showed signs of vigorous re-growthon soil, although the development was slightly delayed compared tounstressed plants, presumably because of the transient exposure tostress. The plants transferred to soil were sprayed with B Basta^(R) toeliminate any false-positives, or any lines where the Basta^(R) markerwas suppressed. All of the Basta^(R)-resistant candidates flowered andset seed. Resistant seedlings were recovered from Superpools 1, 7 and 11on all screens (Table 3-1).

TABLE 3-1 Number of stress-tolerant and Basta^(R) seedlings identifiedon drought surrogate screens. Superpool Promoter 18% PEG 375 mM Mannitol1.5 uM ABA SP1 35S 10 13 11 SP2 35S 0 6 5 SP3 35S 0 2 10 SP4 35S 0 4 0SP5 35S 0 0 5 SP6 35S 0 0 4 SP7 35S 1 4 8 SP8 35S 0 4 1 SP9 32449 0 0 12SP10 32449 0 0 14 SP11 32449 15 3 13 SP12 32449 3 0 6 SP13 32449 0 2 1Qualitative and Quantitative Analysis of 5 Independent EventsRepresenting 358::cDNA 12333678 on PEG, Mannitol and ABA

Seedlings that survived transfer to soil and which wereBasta^(R)-resistant were subjected to PCR and sequencing. At least oneresistant plant in each of the three osmotic screens contained 35S::cDNA12333678 (ME01334). T₂ seeds from the 5 independent transformants thatcontain this clone and that were used in the pooling process were testedfor Basta^(R) resistance and for stress tolerance in the 3 surrogatedrought screens.

To identify two independent events of 35S::cDNA 12333678 showing PEG,mannitol, and ABA resistance, 36 seedlings from each of four events,ME01334-01, 02, 03, and 04 were screened as previously described.Simultaneously, Basta^(R) segregation was assessed to identify eventscontaining a single insert segregating in a 3:1 (R:S) ratio ascalculated by a Chi-square test (Table 3-2). All of the events testedsegregated for a single functional insert.

TABLE 3-2 Basta^(R) segregation for ME01334 individual eventsProbability Event Resistant Sensitive Total of Chi-test* ME01334-01 28 836 0.70031 ME01334-02 22 14 36 0.05429 ME01334-03 31 5 36 0.12366ME01334-04 24 12 36 0.24821 ME01334-5 Insufficient seeds to test*Chi-test to determine whether actual ratio of resistant to sensitivediffers from the expected 3:1 ratio.

Events ME01334-01 and 04 were chosen as the two events because they hadthe strongest and most consistent resistance to PEG, mannitol and ABA.Resistance was observed for ME01334-02 and 03 although not in expectedratios in all three screens (data not shown). The controls were sown thesame day and in the same plate as the individual events. The transgeniccontrol in each of these plates is a segregant, from this ME line oranother ME line being tested, that failed to show resistance to theparticular stress. The PEG (Tables 3-3 and 3-4), mannitol (Tables 3-5and 3-6) and ABA (Tables 3-7 and 3-8) segregation ratios observed forME01334-01 and 01 are consistent with the presence of a single insert asdemonstrated by Chi-Square. This is similar to that observed forBasta^(R) resistance (Table 3-2).

On 18% PEG, the resistant seedlings from these two events show some rootgrowth but they are also green with new leaves emerging at day 14. Themannitol-resistant seedlings showed more root and shoot growth than thePEG resistant seedlings. The resistant seedlings on ABA show the leastamount of growth, and very little root growth relative to the mannitoland PEG screens. The phenotype of the resistant seedlings is unique oneach of the screens.

TABLE 3-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-01 containing 35S::cDNA 12333678 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 26 26.25 0.002 0.922 PEGSensitive 9 8.75 0.007 35 35 0.009

TABLE 3-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-04 containing 35S::cDNA 12333678 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 27 27 0 1.0 PEG Sensitive9 9 0 36 36 0

TABLE 3-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-01 containing 35S::cDNA 12333678 on mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 14 23.25 3.68 0.0001Mannitol Sensitive 17 7.75 11.04 31 31 14.72

TABLE 3-6 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-04 containing 35S::cDNA 12333678 on mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 16 22.5 1.88 0.006Mannitol Sensitive 14 7.5 5.63 30 30 7.51

TABLE 3-7 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-01 containing 35S::cDNA 12333678 on ABA. Probability EventObserved Expected χ² of Chi-Test ABA Resistant 24 24 0 1.0 ABA Sensitive8 8 0 32 32 0

TABLE 3-8 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01334-04 containing 35S::cDNA 12333678 on ABA. Probability EventObserved Expected χ² of Chi-Test ABA Resistant 19 25.5 1.657 0.01 ABASensitive 15 8.5 4.97 34 34 6.627Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon PEG, Mannitol and ABA

Progeny from T₂ plants that were recovered from the three screens andcontained cDNA 12333678 (SP1-A2, SP1-P3, and SP1-M19) were analyzed andalso found to be resistant to PEG, mannitol and ABA indicating thatresistance is transmitted to the next generation. Taken together, 1) theisolation of resistant seedlings containing cDNA 12333678 from all threesurrogate screens for drought, 2) the inheritance of this resistance inthe next generation, and 3) the fact that the progeny from two or moreevents from the original transformation also segregated for resistanceto these osmotic stresses, provides strong evidence that cDNA 12333678when over-expressed provides tolerance to osmotic stress.

This gene is annotated as an alpha/beta hydrolase, a probableacetone-cyanohydrin lyase. Acetone-cyanohydrin lyase is involved in thecatabolism of cyanogenic glycosides.

Table 3-9 provides the results of the consensus sequence (SEQ ID NOs:130-146) analysis based on Ceres cDNA 12333678.

TABLE 3-9 /tmp/Lead-clone26006.aln

23/05/05 plur = 9.500000 -collision -box -noboxcol colbyconsensus

Example 4 Ceres cDNA 12384873

Clone 34419, Ceres cDNA 12384873, encodes a full-length strictosidinesynthase. Ectopic expression of Ceres cDNA 12384873 under the control ofthe CaMV35S promoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol, and abscissic acid (ABA).    -   Continued growth on high PEG, mannitol, and ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12384873

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aT_(i) plasmid containing cDNA 12384873 in the sense orientation relativeto the CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on high PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 12384873 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (ME01490) were screened on high PEG, mannitol andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Qualitative Analysis of 13 Superpools on PEG, Mannitol, and ABA.

Resistant candidates were selected based on increased size when comparedto the largest wild-type control seedlings. All three screens resultedin a decrease in germination for both wildtype and superpools ascompared to seeds on control media. Wild-type seeds that germinated onany of the screens were small and never developed any first leaves evenafter 40 days. To ensure that even slightly tolerant individuals werenot omitted, seedlings that showed any growth and greening whatsoeverwere recovered and transferred to soil for assessment in the T₃generation. All recovered candidates showed signs of vigorous re-growthon soil, although the development was slightly delayed as compared tounstressed plants, presumably because of the transient exposure tostress. The plants transferred to soil were sprayed with Basta^(R) toeliminate any false-positives, or any lines where the Basta^(R) markerwas suppressed. All of the Basta^(R)-resistant candidates flowered andset seed. Resistant seedlings were recovered from Superpools 1, 7 and 11on all screens (Table 4-1).

TABLE 4-1 Number of stress-tolerant and Basta^(R) seedlings identifiedon drought surrogate screens. Superpool Promoter 18% PEG 375 mM Mannitol1.5 uM ABA SP1 35S 10 13 11 SP2 35S 0 6 5 SP3 35S 0 2 10 SP4 35S 0 4 0SP5 35S 0 0 5 SP6 35S 0 0 4 SP7 35S 1 4 8 SP8 35S 0 4 1 SP9 32449 0 0 12SP10 32449 0 0 14 SP11 32449 15 3 13 SP12 32449 3 0 6 SP13 32449 0 2 1Qualitative and Quantitative Analysis of 5 Independent EventsRepresenting 35S::cDNA 12384873 on PEG, Mannitol and ABA

Seedlings that survived transfer to soil and which wereBasta^(R)-resistant were subjected to PCR and sequencing. At least oneresistant plant in each of the three osmotic screens contained 35S::cDNA12384873 (ME01490g. T₂ seeds from the 5 independent transformants thatcontain this clone and that were used in the pooling process were testedfor Basta^(R) resistance and for stress tolerance in the 3 surrogatedrought screens.

To identify two independent events of 35S::cDNA 12384873 showing PEG,mannitol and ABA resistance, 36 seedlings from each of five events,ME01490-01, 02, 03, 04, and 05 were screened as previously described.Simultaneously, Basta^(R) segregation was assessed to identify eventscontaining a single insert segregating in a 3:1 (R:S) ratio ascalculated by a Chi-square test (Table 4-2). Three of the eventssegregated for a single functional insert (-01, -02 and -04). For theother two events one segregated for two independent inserts (-03) andone segregated for a deficiency of Basta^(R) seedlings (-05).

TABLE 4-2 Basta^(R) segregation for ME01490 individual eventsProbability Event Resistant Sensitive Total of Chi-test* ME01490-01* 2412 36 0.24821 ME01490-02* 26 10 36 0.70031 ME01490-03 35 1 36  0.00208**ME01490-04 28 8 36 0.70031 ME01490-05 21 15 36  0.02092** *Chi-test todetermine whether actual ratio of resistant to sensitive differs fromthe expected 3:1 ratio. **Significantly different than a 3:1 (R:S) ratio

Events ME01490-01 and -02 were chosen as the two events because they hadthe strongest and most consistent resistance to PEG, mannitol and ABA.Resistance was observed for ME01490-03, -04, and -05 although not inexpected ratios in all three screens (data not shown). The controls weresown the same day and in the same plate as the individual lines. Thetransgenic control in each of these plates is a segregant, from this MEline or another ME line being tested, that failed to show resistance tothe particular stress. The PEG (Tables 4-3 and 4-4), mannitol (Tables4-5 and 4-6) and ABA (Tables 4-7 and 4-8) segregation ratios observedfor ME01490-01 and -02 are consistent with the presence of singleinsert, as demonstrated by Chi-square. This result is similar to thatobserved for Basta^(R) resistance (Table 4-2).

TABLE 4-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-01 containing 35S::cDNA 12384873 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 29 27 0.148 0.441 PEGSensitive 7 9 0.444 36 36 0.592

TABLE 4-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-02 containing 35S::cDNA 12384873 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 24 24.75 0.0227 0.763 PEGSensitive 9 8.25 0.068 33 33 0.0907

TABLE 4-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-01 containing 35S::cDNA 12384873 on mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 22 26.25 0.688 0.097Mannitol Sensitive 13 8.75 2.06 35 35 2.748

TABLE 4-6 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-02 containing 35S::cDNA 12384873 on mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 20 23.25 0.454 0.178Mannitol Sensitive 11 7.75 1.363 31 31 1.817

TABLE 4-7 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-01 containing 35S::cDNA 12384873 on ABA. Probability EventObserved Expected χ² of Chi-Test ABA Resistant 28 27 0.037 0.7 ABASensitive 8 9 0.148 36 36 1.85

TABLE 4-8 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME01490-02 containing 35S::cDNA 12384873 on ABA. Probability EventObserved Expected χ² of Chi-Test ABA Resistant 23 27 0.593 0.124 ABASensitive 13 9 1.78 36 36 2.373Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon PEG, Mannitol and ABA

Progeny of the T₂ plants that were recovered from the three screens andcontained cDNA 12384873 (SP1-A18, SP1-P14, SP1-M5, SP1-M6 and SP1-M7)were analyzed and also found to be resistant to PEG, mannitol and ABAindicating that resistance is transmitted to the next generation. Takentogether, 1) the isolation of resistant seedlings containing cDNA12384873 from all three surrogate screens for drought, 2) theinheritance of this resistance in the next generation, and 3) the factthat the progeny from two or more events from the originaltransformation also segregated for resistance to these osmotic stresses,provides strong evidence that cDNA 12384873 when over-expressed canprovide tolerance to osmotic stress.

Clone 34419 encodes the first 29 amino acids of a strictosidine synthaseprotein, and then a frame shift results in a novel stretch of 63 aminoacids on the 3′ end of the protein.

Example 5 Ceres cDNA 12659859

Ceres cDNA 12659859 encodes a FAD-linked oxidoreductase family, aprobable berberine bridge enzyme from Arabidopsis thaliana. Ectopicexpression of Ceres cDNA 12659859 under the control of the CaMV35Spromoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol and abscissie acid (ABA).    -   Continued growth on high concentration of PEG, mannitol and ABA.        Generation and Phenotypic Evaluation of T_(I) Lines Containing        35S::cDNA 12659859.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12659859 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasm id vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 12659859 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (SR01010) were screened on high PEG, mannitol, andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Qualitative and Quantitative Analysis of 2 Independent EventsRepresenting 35S::cDNA 12659859 (S1101010) on PEG, Mannitol and ABA

To identify two independent events of 35S::cDNA 12659859 showing PEG,mannitol, and ABA resistance, 36 seedlings from each of three events,SR01010-01, 02, and 03 were screened as previously described. Basta^(R)segregation was assessed to identify lines containing a single insertsegregating in a 3:1 (R:S) ratio as calculated by a Chi-square test(Table 5-1). Two of three T₂ generation events (01 and 03) segregatedfor a single insert although the segregation ratio for -01 is also notdifferent than a 15:1 (R:S) ratio.

TABLE 5-1 Basta^(R) segregation for SR01010 individual eventsProbability Event Resistant Sensitive Total of Chi-test* SR01010-01 32 436 0.05429 SR01010-02 32 3 35  0.0248** SR01010-03 24 12 36 0.24821SR01010-01-1 27 9 36 1     SR01010-03-1 20 13 33 0.05619 *Chi-test todetermine whether actual ratio of resistant to sensitive differs fromthe expected 3:1 ratio. **Significantly different than a 3:1 (R:S) ratio

Lines SR0101-01 and -03 were chosen as the two events because they had astrong and consistent resistance to PEG, mannitol and ABA. Resistancewas observed for SR01010-02 although not in expected ratios in all threescreens (data not shown). The controls were sown the same day and in thesame plate as the individual lines. The PEG (Tables 5-2 and 5-3),mannitol (Tables 5-4 and 5-5) and ABA (Tables 5-6 and 5-7) segregationratios observed for SR01010-01 and -03 are consistent with the presenceof single insert as demonstrated by chi-square, similar to what weobserved for Basta^(R) resistance (Table 5-1).

The progeny from one resistant T₂ plant from each of these two eventswas tested in the T₃ generation in the same manner. Resistance to PEG,mannitol and ABA was also observed in the T₃ generation. Taken together,the segregation of resistant seedlings containing cDNA 12659859 from twoevents on all three drought surrogate screens and the inheritance ofthis resistance in a subsequent generation, provide strong evidence thatcDNA 12659859 when over-expressed can provide tolerance to drought.

TABLE 5-2 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-01T₂ containing 35S::cDNA 12659859 on PEG. Event ObservedExpected χ² Probability PEG Resistant 31 26.25 .860 .064 PEG Sensitive 48.75 2.579 35 35 3.438

TABLE 5-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-03 T₂ containing 35S::cDNA 12659859 on PEG. Event ObservedExpected χ² Probability PEG Resistant 28 27 .037 .700 PEG Sensitive 8 9.111 36 36 .148

TABLE 5-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-01T₂ containing 35S::cDNA 12659859 on mannitol. Event ObservedExpected χ² Probability Mannitol Resistant 25 27 .148 .441 MannitolSensitive 11 9 .444 36 36 .593

TABLE 5-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-3 T₂ containing 35S::cDNA 12659859 on mannitol. Event ObservedExpected χ² Probability Mannitol Resistant 21 20.25 .028 .739 MannitolSensitive 6 6.75 .083 27 27 .111

TABLE 5-6 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-01T₂ containing 35S::cDNA 12659859 on ABA. Event ObservedExpected χ² Probability ABA Resistant 30 27 .333 .248 ABA Sensitive 6 91 36 36 1.333

TABLE 5-7 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01010-03 T₂ containing 35S::cDNA 12659859 on ABA. Event ObservedExpected χ² Probability ABA Resistant 27 27 0 1.0 ABA Sensitive 9 9 0 3636 0

Table 5-8 provides the results of the consensus sequence (SEQ ID NOs:147-177) analysis based on Ceres cDNA 12659859.

TABLE 5-8 /tmp/Lead•cDNA12659859.oln

23/05/05 plur = 5.000000 -collision -box -noboxcol colbyconsensus

Example 6 Ceres cDNA 12723147

Ceres cDNA 12723147 encodes an Arabidopsis putative aldo/keto reductase.Ectopic expression of Ceres cDNA 12723147 under the control of theCaMV35S promoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol and abscissic acid (ABA).    -   Continued growth on high concentration of PEG, mannitol and ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12723147.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12723147 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasm id vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T_(I) generation.No positive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 12723147 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (SR01013) were screened on high PEG, mannitol, andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Qualitative and Quantitative Analysis of 2 Independent EventsRepresenting 35S::cDNA 12659859 (SR01010) on PEG, Mannitol and ABA

To identify two independent events of 35S::cDNA 12659859 showing PEG,mannitol, and ABA resistance, 36 seedlings from each of two events,SR01013-01 and -02 were screened as previously described. Basta^(R)segregation was assessed to verify that the lines contained a singleinsert segregating in a 3:1 (R:S) ratio as calculated by a chi-squaretest (Table 6-1). Both lines (01 and 02) segregated for a single insertin the T₂ generation (Table 1)

TABLE 6-1 Basta^(R) segregation for SR01013 individual eventsProbability Event Resistant Sensitive Total of Chi-test* SR01013-01 30 535 0.14323  SR01013-02 30 6 36 0.24821  SR01013-01-3 34 1 36 0.00248**SR01013-02-2 32 0 32 0.00109** *Chi-test to determine whether actualratio of resistant to sensitive differs from the expected 3:1 ratio.**Significantly different than a 3:1 (R:S) ratio

Lines SR01013-01 and -02 were chosen as the two events because they hada strong and consistent resistance to PEG, mannitol and ABA. Thecontrols were sown the same day and in the same plate as the individuallines. The PEG (Tables 6-2 and 6-3), mannitol (Tables 6-4 and 6-5) andABA (Tables 6-6 and 6-7) segregation ratios observed for SR01013-01 and-02 are consistent with the presence of single insert as demonstrated bychi-square, similar to what we observed for Basta^(R) resistance (Table6-1).

The progeny from one resistant T₂ plant from each of these two eventswere tested in the same manner as the T₂. Resistance to PEG, mannitoland ABA was also observed in the T₃ generation. Taken together, thesegregation of resistant seedlings containing cDNA 12723147 from twoevents on all three drought surrogate screens and the inheritance ofthis resistance in a subsequent generation, provide strong evidence thatcDNA 12723147 when over-expressed can provide tolerance to drought.

TABLE 6-2 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-01T₂ containing 35S::cDNA 12723147 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 22 27 0.926 0.054 PEGSensitive 14 9 2.778 36 36 3.704

TABLE 6-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T₂ containing 35S::cDNA 12723147 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 26 27 0.037 .700 PEGSensitive 10 9 .111 36 36 .148

TABLE 6-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-01 T₂ containing 35S::cDNA 12723147 on mannitol. ProbabilityEvent Observed Expected χ² of Chi-Test Mannitol Resistant 28 27 .037.700 Mannitol Sensitive 8 9 .111 36 36 .148

TABLE 6-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T₂ containing 35S::cDNA 12723147 on mannitol. ProbabilityEvent Observed Expected χ² of Chi-Test Mannitol Resistant 18 27 3 .0005Mannitol Sensitive 18 9 9 36 36 12

TABLE 6-6 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T₂ containing 35S::cDNA 12723147 on ABA. Event ObservedExpected χ² Probability ABA Resistant 13 24 5.042 7.098 ABA Sensitive 198 15.125 32 32 20.167

TABLE 6-7 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01013-02 T₂ containing 35S::cDNA 12723147 on ABA. Event ObservedExpected χ² Probability ABA Resistant 13 24 5.042 7.098 ABA Sensitive 198 15.125 32 32 20.167Table 6-8 provides the results of the consensus sequence (SEQ ID NOs:178-200) analysis based on Ceres cDNA 12723147.

TABLE 6-8 /tmp/Lead•cDNA.oln

23/05/05 plur = 16.500000 -collision -box -noboxcol colbyconsensus

Example 7 Ceres cDNA 13488750

Clone 125039, Ceres cDNA 13488750, encodes a full-length putativeadenylylsulfate (APS) kinase from Arabidopsis thaliana. Ectopicexpression of Ceres cDNA 13488750 under the control of the CaMV35Spromoter induces the following phenotypes:

-   -   Continued growth under high heat conditions.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 13488750.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 13488750 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T_(I) generation.No negative phenotypes were observed in the T₁ plants although three ofthe T1 lines produced a small rosette (ME02526-01, 02 and 05). T₂ and T₃lines of events ME02526-01 and ME02526-05 did not show the small rosettephenotype.

Screens of Masterpools for Heat Tolerance Via Heat Shock In Vitro.

Seeds from 100 masterpools from the CaMV35S or 32449 over-expressionlines were tested for heat tolerance in vitro as described above.

Once cDNA 13488750 was identified in tolerant plants from the screen,five individual T₂ events containing this cDNA (ME02526) were screenedon soil as described above to identify events with the resistancephenotype.

Qualitative Analysis of the T₂ Masterpool of cDNA 13488750 Plants HeatShocked on Plates

Visual phenotyping of the masterpool containing cDNA 13488750 (ME02526)on agar or soil showed no visible alterations in phenotype (data notshown). After heat-shock at 15 days of age, the ME02526 masterpoolshowed greater heat recovery as compared to the wild-type control andother transgenic masterpools. Assessment was a measure of “greenness” aswell as continued growth at 23° C. after heat shock at 45° C. forbetween 5 and 8 hours. Immediately alter the heat shock stress, theextent of stress-induced damage in the control and ME02526 masterpoolappeared comparable. The leaves and cotyledons were wilted and droopyalthough still green. However, after 4 days of recovery, 2 of 10 plantsin the ME02526 masterpool had completely recovered and were growingagain. Other seedlings showed some recovery as measured by greennesscompared to the wild-type control.

Qualitative and Quantitative Analysis of Individual T₂ Events of cDNA13488750 Under Continual Heat Treatment on Soil

Five independent events of ME02526 were tested on soil as describedabove. Two of the events (ME02526-04 and -05) showed heat resistanceafter continual growth at 36° C. Heat resistance was noted as decreasedchlorosis compared to wild-type. Segregation frequencies of thetransgene under test suggest that these two events contain a singleinsert, as calculated by a chi-square test (Tables 7-1, 7-2 and 7-3).Ten and 11 plants from events 04 and 05, respectively, showed continuedvigor and decreased chlorosis after continuous heat treatment comparedto wild-type controls.

TABLE 7-1 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-04 and -05 containing 35S::cDNA 13488750 on Finale. ProbabilityEvent Observed Expected χ² of Chi-Test ME02526-04 Finale 29 27 0.5930.44 Resistant ME02526-04 Finale 7 9 Sensitive 36 36 ME02526-05 Finale27 27 0 1 Resistant ME02526-05 Finale 9 9 Sensitive 36 36

TABLE 7-2 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-04 containing 35S::cDNA 13488750 for thermo tolerance (continualgrowth at 36° C. on soil). Probability Event Observed Expected χ² ofChi-Test Heat Tolerant 10 11.25 0.556 0.456 Heat Sensitive 5 3.75 15 15

TABLE 7-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-05 containing 35S::cDNA 13488750 for thermo tolerance (continualgrowth at 36° C. on soil). Probability Event Observed Expected χ² ofChi-Test Heat Tolerant 11 11.25 0.022 0.881 Heat Sensitive 4 3.75 15 15

The plants that survive the heat treatment show premature bolting andreduced fecundity but much less so than the control plants. Control andME02526 plants bolt after only 4 days of exposure to 36° C. (11-day oldplants), but were more vigorous and less chlorotic than the controls andthe height and branch number was comparable to those of wild-type (datanot shown). Heat-tolerant lines all showed seed abortion and reducedfecundity (data not shown). Seed abortion and reduced silique size wasalso prevalent in all wild-type controls (data not shown). Events 04 and05, which had a thermo tolerant phenotype in the T₂ generation, wereevaluated in greater detail in the T₃ generation for heat resistance andfecundity after prolonged heat stress on MS plates.

Qualitative and Quantitative Analysis of Individual T₃ Events Under 36°C. Heat Treatment on Plates

Seeds from five individuals of the T₃ generation for ME02526-04 and -05lines and controls were sterilized, stratified and germinated for 7 daysat 23° C. prior to exposure to 36° C. heat stress. The events wereevaluated for heat resistance to prolonged heat stress.

The thermo tolerant phenotype became apparent after 15 days of 36° C.treatment. T₃ progeny from ME02526-04 (Table 7-4) and ME02526-05 (Table7-5) were found to segregate in the expected 3:1 ratio for the thermotolerant phenotype.

TABLE 7-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-04 containing 35S::cDNA 13488750 for thermo tolerance (continualgrowth at 36° C. Probability Event Observed Expected χ² of Chi-Test HeatTolerant 14 14.25 0.017 0.89 Heat Sensitive 5 4.75 19 19

TABLE 7-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME02526-05 containing 35S::cDNA 13488750 for thermo tolerance (continualgrowth at 36° C. Probability Event Observed Expected χ² of Chi-Test HeatTolerant 15 14.25 0.157 0.69 Heat Sensitive 4 4.75 19 19Qualitative Analysis of Individual T₃ Events Heat Shocked on Plates forDifferentiation of Natural Acquired Thermotolerance

Plants acquire thermotolerance to lethal high temperatures such as 45°C. if previously exposed to moderately high temperature or if thetemperature is raised gradually to an otherwise lethal temperature(Vierling, 1991). To ascertain whether the thermotolerance observed inthe T₃ generation is due to some naturally acquired thermotoleranceimparted by heat exposure of the T₂ parent plant (ME2526-04 and 05),thermotolerance was assessed by comparing pre-heat treated wild-type andtransgenic controls and unheated wild-type controls. T₃ events ofME02526-04 and 05 were heat shocked for 4 hours as described above.ME02526-04 and -05 were able to stay greener longer than both pre-heattreated controls and un-heat treated controls. However, both ME02526lines and all controls (wild-type and transgenic) failed to elongate andthrive after heat treatment at 45° C. and eventually all died. Un-heattreated wild-type control became chlorotic faster (1-day aftertreatment) than pre-heat treated wildtype control and pre-heat treatedtransgenic control suggesting that there is some natural acquiredthermotolerance that occurs that is not correlated with theover-expression of 35S::cDNA 13488750. Even with exposure to this lethaltemperature, ME02526 was greener after 7 days than controls, indicatingthat ME02526 has a thermotolerance phenotype that is unrelated to thenatural mechanisms of acquired thermotolerance.

Table 7-6 provides the results of the consensus sequence (SEQ ID NOs:201-214) analysis based on Ceres cDNA 13488750.

TABLE 7-6 /tmp/Lead•clone125039.oln

23/05/05 plur = 3.000000 -collision -box -noboxcol colbyconsensus

Example 8 Ceres cDNA 13489782

Clone 10044, Ceres cDNA 13489782, encodes a full-length putative114-amino acid hypothetical protein from Arabidopsis thaliana. Ectopicexpression of Ceres cDNA 13489782 under the control of the 32449promoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG)        and abscissic acid (ABA) and    -   Continued growth on high concentrations of PEG and ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        32449::cDNA 13489782

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 13489782 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 13489782 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (ME00446) were screened on high PEG, mannitol, andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Qualitative Analysis of Superpools on PEG, Mannitol, and ABA.

Resistant candidates were selected based on increased size when comparedto the largest wild-type control seedlings. All three screens resultedin a decrease in germination for both wildtype and superpools ascompared to seeds on control media. Wild-type seeds that germinated onany of the screens were small and never developed any first leaves evenafter 40 days. To ensure that even slightly tolerant individuals werenot omitted, seedlings that showed any growth and greening whatsoeverwere recovered and transferred to soil for assessment in the T₃generation. All recovered candidates showed signs of vigorous re-growthon soil, although the development was slightly delayed as compared tounstressed plants, presumably because of the transient exposure tostress. The plants transferred to soil were sprayed with Basta toeliminate any false-positives, or any lines where the Basta^(R) markerwas suppressed. All of the Basta-resistant candidates flowered and setseed. Resistant seedlings were recovered from Superpools 1, 7 and 11 onall screens (Table 8-1).

TABLE 8-1 Number of stress-tolerant and Basta^(R) seedlings identifiedon drought surrogate screens. Superpool Promoter 18% PEG 375 mM Mannitol1.5 uM ABA SP1 35S 10 13 11 SP2 35S 0 6 5 SP3 35S 0 2 10 SP4 35S 0 4 0SP5 35S 0 0 5 SP6 35S 0 0 4 SP7 35S 1 4 8 SP8 35S 0 4 1 SP9 32449 0 0 12SP10 32449 0 0 14 SP11 32449 15 3 13 SP12 32449 3 0 6 SP13 32449 0 2 1

We obtained sequence from 3, 3, and 13 plants that were both Basta^(R)and resistant to PEG, mannitol or ABA, respectively. For each of thethree surrogate drought screens, one or more plants contained the32449::clone 10044 (ME00446). The probability of finding a plantcontaining this cDNA at random in all three screens is 0.03×0.03×0.03.

Qualitative and Quantitative Analysis of 6 Independent EventsRepresenting 32449::cDNA 13489782 on PEG, Mannitol and ABA

To identify independent events of 32449::cDNA 13489782 showing PEG,mannitol and ABA resistance, 36 seedlings from each of six events,ME00446-01, 02, 03, 04, 05 and 06 were screened as previously described.Simultaneously, Basta^(R) segregation was assessed to identify linescontaining a single insert segregating in a 3:1 (R:S) ratio ascalculated by a Chi-square test (Table 8-2). All of the lines segregatedfor a single functional insert.

TABLE 8-2 Basta^(R) segregation for 6 events of ME00446 Did Not Resis-Sensi- Probability ME Line Assessment Germinate tant tive Total ofChi-test 00446-01 Oct. 30, 2003 1 28 7 35 0.49452 00446-02 Oct. 30, 20030 28 8 36 0.70031 00446-03 Oct. 30, 2003 1 28 7 35 0.49452 00446-04 Oct.30, 2003 1 27 8 35 0.7697 00446-05 Oct. 30, 2003 2 24 10 34 0.5524500446-06 Oct. 30, 2003 0 25 11 36 0.44142 *Chi-test to determine whetheractual ratio of resistant to sensitive differs from the expected 3:1ratio.

Events ME00446-02 and 04 were chosen as the two events for furtheranalysis because they had the strongest and most consistent resistanceto PEG and ABA. None of the lines showed mannitol resistance at 375 mMconcentration. The controls were sown the same day and in the same plateas the individual lines. The PEG (Tables 8-3 and 8-4) and ABA (Tables8-5 and 8-6) segregation ratios observed for ME00446-02 and -04 areconsistent with the presence of single insert as demonstrated by theChi-Square test. This is similar to that observed for Basta resistance(Table 8-2).

Despite the fact that this line was isolated from all three screens, itwas subsequently concluded that it could not be considered mannitolresistant. This is likely due to an overly stringent mannitolconcentration. In a superpool screen setting, the seedlings are moredensely grown than in an individual line setting. This means that in asuperpool screen, there is a lower effective concentration of mannitol.When putative tolerant plant from a superpool is tested as an individualline, the effective concentration it is grown on is actually higher. Inthe case of ME00446, this difference was enough to invalidate it asmannitol tolerant. In fact, a resistant plant to mannitol was isolatedfrom superpool 11 that corresponds to clone 10044.

TABLE 8-3 Chi-square analysis assuming a 3:1(R:S) ratio for progeny ofME00446-02 containing 32449::clone 10044 on PEG. Probability EventObserved Expected χ² of χ² PEG Resistant 30 26.95 0.3452 0.2557 PEGSensitive 5 7.7 0.9468 Total 35 35 1.292

TABLE 8-4 Chi-square analysis assuming a 3:1(R:S) ratio for progeny ofME00446-04 containing 32449::clone 10044 on PEG. Probability EventObserved Expected χ² of χ² PEG Resistant 26 27.77 0.113 0.482 PEGSensitive 10 8.23 0.3813 Total 36 36 0.4943

TABLE 8-5 Chi-square analysis assuming a 3:1(R:S) ratio for progeny ofME00446-02 containing 32449::clone 10044 on ABA. Probability EventObserved Expected χ² of χ² ABA Resistant 31 27 0.5926 0.1237 ABASensitive 5 9 1.778 Total 36 36 2.370

TABLE 8-6 Chi-square analysis assuming a 3:1(R:S) ratio for progeny ofME00446-04 containing 32449::clone 10044 on ABA. Probability EventObserved Expected χ² of χ² ABA Resistant 31 27 0.5926 0.1237 ABASensitive 5 9 1.778 Total 36 36 2.370Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon PEG, Mannitol and ABA

Progeny from T₂ plants that were recovered from the three screens andcontaining clone 10044 (SP11-M13 and SP11-P5) were found to be resistantto PEG and ABA. Taken together, 1) the isolation of resistant seedlingscontaining clone 10044 from all three surrogate screens for drought, 2)the inheritance of this resistance in the next generation, and 3) thefact that the progeny from two or more events from the originaltransformation also segregated for resistance to these osmotic stresses,provide strong evidence that clone 10044 when over-expressed providesresistance to osmotic and dehydration stress.

Table 8-7 provides the results of the consensus sequence (SEQ ID NOs:215-222) analysis based on Ceres cDNA 13489782.

TABLE 8-7 /tmp/Lead•clone10044.oln

23/05/05 plur = 2.000000 -collision -box -noboxcol colbyconsensus

Example 9 Ceres cDNA 13486759

Clone 10987, corresponding Ceres cDNA 13486759, encodes an Arabidopsis251-amino acid expressed protein. Ectopic expression of clone 10987under the control of the CaMV35S promoter induces the followingphenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol, and abscissic acid (ABA), and    -   Continued growth on high concentrations of PEG, mannitol, and        ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 13486759

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 13486759 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol and ABA as Surrogate Screensfor Drought Tolerance

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 13486759 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (ME03316) were screened on high PEG, mannitol, andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Qualitative Analysis of 13 Superpools on PEG, Mannitol and ABA

Resistant candidates were selected based on increased size when comparedto the largest wild-type control seedlings. All three screens resultedin a decrease in germination for both wildtype and superpools ascompared to seeds on control media. Wild-type seeds that germinated onany of the screens were small and never developed any first leaves evenafter 40 days. To ensure that even slightly tolerant individuals werenot omitted, seedlings that showed any growth and greening whatsoeverwere recovered and transferred to soil for assessment in the T₃generation. All recovered candidates showed signs of vigorous re-growthon soil, although the development was slightly delayed as compared tounstressed plants, presumably because of the transient exposure tostress. The plants transferred to soil were sprayed with Basta toeliminate any false-positives, or any lines where the Basta^(R) markerwas suppressed. All of the Basta-resistant candidates flowered and setseed. Resistant seedlings were recovered from Superpools 1, 7 and 11 onall screens (Table 9-1).

TABLE 9-1 Number of stress-tolerant and Basta^(R) seedlings identifiedon drought surrogate screens. Superpool Promoter 18% PEG 375 mM Mannitol1.5 uM ABA SP1 35S 10 13 11 SP2 35S 0 6 5 SP3 35S 0 2 10 SP4 35S 0 4 0SP5 35S 0 0 5 SP6 35S 0 0 4 SP7 35S 1 4 8 SP8 35S 0 4 1 SP9 32449 0 0 12SP10 32449 0 0 14 SP11 32449 15 3 13 SP12 32449 3 0 6 SP13 32449 0 2 1

We obtained sequence from 3, 3, and 13 plants from Superpool 11 thatwere both Basta^(R)-resistant and resistant to PEG, mannitol or ABA,respectively. For each of the three osmotic screens, one or more plantscontained the 35S::clone 10987 (ME03316), which made it a good candidatefor further testing. The probability of finding a plant containing thisclone 10987 at random in all three screens is 0.03×0.03.

Qualitative and Quantitative Analysis of 5 Independent EventsRepresenting 35S::cDNA 13486759 on PEG, Mannitol and ABA

To identify independent events of 35S::cDNA 13486759 showing PEG,mannitol and ABA resistance, 36 seedlings from each of five events,ME03316-01, -02, -03, -04, and -05 were screened as previouslydescribed. Simultaneously, Basta^(R) segregation was assessed toidentify events containing a single insert segregating in a 3:1 (R:S)ratio as calculated by a chi-square test (Table 9-2). All of the eventssegregated for a single functional insert. ME03316-02 could besegregating for two linked or unlinked inserts but the ratios on thesurrogate drought screens indicate it is likely to be a single insert.

TABLE 9-2 Basta^(R) segregation for 5 individual events ProbabilityEvent Resistant Sensitive Total of Chi-test ME03316-01 23 13 36 0.12366ME03316-02 32 4 36 0.05429 ME03316-03 30 6 36 0.24821 ME03316-04 25 1136 0.44142 ME03316-05 29 7 36 0.44142 *Chi-test to determine whetheractual ratio of resistant to sensitive differs from the expected 3:1ratio.

Lines ME03316-01 and 02 were chosen as the two events for furtheranalysis because they had the strongest and most consistent resistanceto PEG, mannitol and ABA. Resistance was observed for ME03316-03, 04,and 05 although not in expected ratios in all three screens (data notshown). The controls were sown the same day and on the same plate as theindividual lines. The PEG (Tables 9-3 and 9-4), mannitol (Tables 9-5 and9-6) and ABA (Table 9-7) segregation ratios observed are consistent withthe presence of a single insert as demonstrated by chi-square.ME03316-02 seedlings on ABA (Table 9-8) appear to be segregating for twoinserts which is still consistent with the ratio observed on Basta^(R).Both events segregate for a deficiency of resistant seedlings onmannitol.

TABLE 9-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME03316-01 containing 35S::clone 10987 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 22 27 0.93 0.0543 PEGSensitive 14 9 2.78 Total 36 36 3.7

TABLE 9-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME03316-02 containing 35S::clone 10987 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 23 26.25 0.4024 0.2046PEG Sensitive 12 8.75 1.2071 Total 35 35 1.610

TABLE 9-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME03316-01 containing 35S::clone 10987 on Mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 19 25.5 1.657 0.01Mannitol Sensitive 15 8.5 4.971 Total 34 34 6.63

TABLE 9-6 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME03316-02 containing 35S::clone 10987 on Mannitol. Probability EventObserved Expected χ² of Chi-Test Mannitol Resistant 18 26.25 2.5930.0013 Mannitol Sensitive 17 8.75 7.779 Total 35 35 10.371

TABLE 9-7 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME03316-01 containing 35S::clone 10987 on ABA. Probability EventObserved Expected χ² of Chi-Test ABA Resistant 27 25.5 0.0882 0.5525 ABASensitive 7 8.5 0.2647 Total 34 34 0.3529

TABLE 9-8 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofME03316-02 containing 35S::clone 10987 on ABA. Probability EventObserved Expected χ² of Chi-Test ABA Resistant 32 24.75 2.124 0.0036 ABASensitive 1 8.25 6.371 Total 33 33 8.495Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon PEG, Mannitol and ABA

Progeny from T₂ plants that were recovered from the three screens andcontaining clone 10987 (SP11-A15, SP11-A16, SP11-P2, and SP11-M10) werefound to be resistant to PEG, mannitol and ABA.

On PEG, the progeny of SP11-M10 segregated for a deficiency of resistantseedlings similar to the deficiency that noted for the T2 seedlings inTables 9-5 and 9-6. A deficiency of resistant seedlings is also notedfor the progeny of SP11P2 on PEG.

Taken together, 1) the isolation of resistant seedlings containing clone10987 from all three surrogate screens for drought, 2) the inheritanceof this resistance in the next generation, and 3) the fact that theprogeny from two or more events from the original transformation alsosegregated for resistance to these osmotic stresses, these findingsprovide strong evidence that clone 10987 when over-expressed can providetolerance to osmotic stresses.

Example 10 Ceres cDNA 13500101

Clone 17206, corresponding to Ceres cDNA 13500101, encodes a putativestrictosidine synthase. Ectopic expression of cDNA 13500101 under thecontrol of the CaMV35S promoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG)        and mannitol.    -   Continued growth on high concentrations of PEG and mannitol.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 13500101.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 13500101 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 13500101 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (SR01000) were screened on high PEG, mannitol, andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Qualitative and Quantitative Analysis of 2 Independent EventsRepresenting 35S::cDNA 13500101 (SR01000) on PEG, Mannitol and ABA

To identify two independent events of 35S::clone 17206 showing PEG,mannitol, and ABA resistance, 36 seedlings from each of three events,SR01000-01, 02 and 03 were screened as previously described. Bastasegregation was assessed to verify that the lines contained a singleinsert segregating in a 3:1 (R:S) ratio as calculated by a chi-squaretest (Table 1). Two lines (-01 and -02) segregated for a single insert(Table 1).

TABLE 10-1 Basta^(R) segregation for SR01000 individual eventsProbability Event Resistant Sensitive Total of Chi-test SR01000-01 29 736 0.44142 SR01000-02 23 13 36 0.12366 SR01000-03 35 0 35 0.00064SR01000-01-01 35 0 35 0.00064 SR01000-02-01 27 9 36 1 SR01000-03-01 36 036 0.00053 *Chi-test to determine whether actual ratio of resistant tosensitive differs from the expected 3:1 ratio.

Testing of the progeny from the T₂ resistant plants on the 3 surrogatedrought screens showed that fines SR01000-01 and 02 had a strong andconsistent resistance to PEG, mannitol, but not to ABA. These werechosen as the two events for further analysis. The controls were sownthe same day and in the same plate as the individual lines. The PEG(Tables 10-2 and 10-3), and mannitol (Tables 10-4 and 10-5) segregationratios observed for SR01000-01 and 02 are consistent with the presence aof single insert as demonstrated by chi-square (Table 10-1).

TABLE 10-2 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01000-01 T₂ containing 35S::cDNA 13500101 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 26 26.25 0.002 0.922 PEGSensitive 9 8.75 0.007 35 35 0.009

TABLE 10-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01000-02 T₂ containing 35S::cDNA 13500101 on PEG. Probability EventObserved Expected χ² of Chi-Test PEG Resistant 27 24.75 0.205 0.366 PEGSensitive 6 8.25 0.614 33 33 0.818

TABLE 10-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01000-01 T₂ containing 35S::cDNA 13500101 on mannitol. ProbabilityEvent Observed Expected χ² of Chi-Test Mannitol Resistant 28 27 0.0370.700 Mannitol Sensitive 8 9 0.111 36 36 0.148

TABLE 10-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01000-02 T₂ containing 35S::cDNA 13500101 on mannitol. ProbabilityEvent Observed Expected χ² of Chi-Test Mannitol Resistant 30 27 0.3330.248 Mannitol Sensitive 6 9 1.000 36 36 1.333Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon PEG, Mannitol and ABA

The progeny from one resistant T₂ plant from each of these two eventswere tested in the same manner as the T₂. Resistance to PEG and mannitolpersisted in the second generation. Taken together, 1) the isolation ofresistant seedlings containing clone 17026 from two of the surrogatescreens for drought (PEG and mannitol), 2) the inheritance of thisresistance in the next generation, and 3) the fact that the progeny fromtwo or more events from the original transformation also segregated forresistance to these osmotic stresses, these findings provide strongevidence that clone 17026 when over-expressed can provide tolerance toosmotic stresses.

Table 10-6 provides the results of the consensus sequence (SEQ ID NOs:223- 240) analysis based on Ceres cDNA 13500101.

TABLE 10-6 /tmp/Lead•clone17206.oln

23/05/05 plur = 5.500000 -collision -box -noboxcol colbyconsensus

Example 11 Ceres cDNA 13509011 (12357529)

Clone 104691, corresponding to Ceres cDNA 13509011 (12357529), encodes aprobable strictosidine synthase enzyme. Ectopic expression of cDNA13509011 under the control of the CaMV35S promoter induces the followingphenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG)        and mannitol.    -   Continued growth on high concentration of PEG and mannitol,        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 13509011.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12334963 in the sense orientation relative tothe CaMV35S constitutive promoter. The T_(i) plasmid vector used forthis construct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol, and ABA as SurrogateScreens for Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on 3 droughtsurrogate screens (high concentrations of PEG, mannitol, and ABA) asdescribed above. T₃ seeds were collected from the resistant plants andanalyzed for resistance on all three surrogate drought screens.

Once cDNA 13509011 was identified in resistant plants from each of thethree surrogate drought screens, the five individual T₂ eventscontaining this cDNA (SR01002) were screened on high PEG, mannitol, andABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Qualitative and Quantitative Analysis of 2 Independent EventsRepresenting 35S::clone 104691 (SR01002) on PEG, Mannitol and ABA

To identify two independent events of 35S::clone 104691 showing PEG,mannitol and ABA resistance, 36 seedlings from each of three events,SR01002-01, 02, and 03 were screened as previously described.Simultaneously, Basta segregation was assessed to identify linescontaining a single insert segregating in a 3:1 (R:S) ratio ascalculated by a chi-square test (Table 1). Two lines (01 and 03)segregated for a single insert.

TABLE 11-1 Basta segregation for SR01002 individual events ProbabilityEvent Resistant Sensitive Total of Chi-test SR01002-01 26 10 36 0.70031SR01002-02 23 8 31 0.91741 SR01002-03 28 8 36 0.70031 SR01002-01-1 36 036 0.00053 SR01002-02-1 28 8 36 0.70031 SR01002-03-1 25 11 36 0.44142*Chi-test to determine whether actual ratio of resistant to sensitivediffers from the expected 3:1 ratio.

Testing of the progeny from the resistant T₂ plants on the 3 surrogatedrought screens showed that lines SR01002-01 and 03 had a strong andconsistent resistance to PEG, mannitol, but not to ABA. These werechosen as the two events for further analysis. The controls were sownthe same day and in the same plate as the individual lines. The PEG(Tables 11-2 and 11-3), and mannitol (Tables 11-4 and 11-5) segregationratios observed for SR01002-01 and 03 are consistent with the presenceof a single insert as demonstrated by chi-square. This is similar tothat observed for Basta resistance (Table 11-1).

TABLE 11-2 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01002-01 T₂ containing 35S::clone 104691 on PEG. Event ObservedExpected χ² Probability PEG Resistant 22 24.75 0.306 0.269 PEG Sensitive11 8.25 0.917 33 33 1.222

TABLE 11-3 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01002-03 T₂ containing 35S::clone 104691 on PEG. Event ObservedExpected χ² Probability PEG Resistant 24 25.5 0.088 0.552 PEG Sensitive10 8.5 0.265 34 34 0.353

TABLE 11-4 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01002-01 T₂ containing 35S::clone 104691 on mannitol. Event ObservedExpected χ² Probability Mannitol Resistant 30 27 0.333 0.248 MannitolSensitive 6 9 1.000 36 36 1.333

TABLE 11-5 Chi-square analysis assuming a 3:1 (R:S) ratio for progeny ofSR01002-03 T₂ containing 35S::clone 104691 on mannitol. Event ObservedExpected χ² Probability Mannitol Resistant 32 27 0.926 0.054 MannitolSensitive 4 9 2.78 36 36 3.70Qualitative and Quantitative Analysis of Progeny of T₂ Plants Isolatedon PEG, Mannitol, and ABA Screens.

The progeny from one resistant T₂ plant from each of these two eventswas tested in the T3 generation in the same manner. Resistance to PEGand mannitol persisted into the next generation. Taken together, 1) theisolation of resistant seedlings containing clone 104691 from two of thesurrogate screens for drought (PEG and mannitol), 2) the inheritance ofthis resistance in the next generation, and 3) the fact that the progenyfrom two or more events from the original transformation also segregatedfor resistance to these surrogate drought conditions, these findingsprovide strong evidence that clone 104691 when over-expressed canprovide tolerance to osmotic stresses.

Table 11-6 provides the results of the consensus sequence (SEQ ID NOs:241-262) analysis based on Ceres cDNA 13509011 (12357529).

TABLE 11-6 /tmp/Lead•clone104691.oln

23/05/05 plur = 6.500000 -collision -box -noboxcol colbyconsensus

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

What is claimed is:
 1. A plant, plant cell, plant material or seed of aplant which comprises a nucleic acid molecule comprising: a) a firstnucleic acid having a nucleotide sequence which encodes an amino acidsequence exhibiting at least 95% sequence identity to SEQ ID NO: 11, ora first nucleic acid having the nucleotide sequence of SEQ ID NO:10which encodes SEQ ID NO: 11; and (b) a second nucleic acid comprising aregulatory sequence capable of causing transcription and/or translationof said first nucleic acid, wherein said first and second nucleic acidsare operably linked and heterologous to each other, and wherein saidplant has improved water use efficiency characteristics as compared to awild-type plant cultivated under the same conditions.
 2. A transgenicplant having a gene construct comprising a nucleic acid encoding a wateruse efficiency component operably linked to a plant promoter so that thewater use efficiency component is ectopically overexpressed in thetransgenic plant, and the transgenic plant exhibits: i) faster rate ofgrowth, ii) greater fresh or dry weight at maturation, iii) greaterfruit or seed yield, iv) higher tolerance to heat, v) higher toleranceto drought, vi) higher tolerance to cold, vii) greater germination rate,viii) reduced water consumption, or ix) greater tolerance to flood thana progenitor plant which does not contain the gene construct, when thetransgenic plant and the progenitor plant are cultivated under identicalenvironmental conditions, wherein the water use efficiency component isa nucleic acid molecule encoding a polypeptide having at least 95%identity to SEQ ID NO:11, and wherein said nucleic acid encoding a wateruse efficiency component is heterologous to said plant promoter.
 3. Theplant, plant cell, plant material or seed of a plant according to claim1, wherein said first nucleic acid has a nucleotide sequence whichencodes the amino acid sequence of SEQ ID NO:
 11. 4. The plant, plantcell, plant material or seed of a plant according to claim 1, whereinsaid first nucleic acid sequence has the sequence of SEQ ID NO: 10 whichencodes SEQ ID NO:
 11. 5. The transgenic plant according to claim 2,wherein said nucleic acid molecule encoding said polypeptide has theamino acid sequence of SEQ ID NO:
 11. 6. The transgenic plant accordingto claim 2, wherein said nucleic acid molecule has the nucleotidesequence of SEQ ID NO: 10 which encodes a polypeptide having the aminoacid sequence of SEQ ID NO: 11.